Composition for skin

Abstract

To provide a method for promoting skin wound healing and a composition for use in promoting skin wound healing; to provide a method or composition useful for preventing or improving ulcers / bedsores; to provide a method for inhibiting skin aging and a composition for use in inhibiting skin aging; to provide a composition useful for preventing or improving skin disorders caused by anticancer agents, and to further provide a method for enhancing the regenerative capacity of epidermal stem cells and a composition for enhancing the regenerative capacity of epidermal stem cells.SOLUTION: By using a composition containing, as an active ingredient, a substance selected from the group consisting of a substance that induces or maintains the expression of COL17A1 in cells, a substance that suppresses degradation of COL17A1 in cells, a substance that promotes the competitive amplification of epidermal stem cells, a substance that suppresses genomic stress or oxidative stress in cells, and a substance that suppresses DNA damage in cells, promotion of skin wound healing, protection against anticancer drugs, suppression of skin aging, and enhancement of the ability to regenerate epidermal stem cells can be achieved.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a composition that has beneficial effects on skin. In particular, the present invention relates to a composition that has the effect of promoting skin wound healing, inhibiting skin aging, and / or enhancing the regenerative ability of epidermal stem cells. More particularly, the present invention relates to the fields of pharmaceutical compositions, cosmetic compositions, and beauty supplements. [Background technology]

[0002] Aging of animal organs is a multi-step process that results in their structural and functional decline. The exact fate of exhausted / stressed cells is not known in detail, although cell senescence and / or stem cell exhaustion have been implicated as cellular dynamics. In general, the in vivo dynamics of cells that compose tissues / organs during aging are largely unknown. Previous studies on mammalian hair follicles (mini-organs of the skin) have demonstrated the central role of stem cell aging in the progression of hair follicle aging and the existence of a "stem cell-centric organ aging program" that causes aging-related hair follicle miniaturization leading to age-related hair thinning and graying (Non-Patent Document 1, Patent Document 2). This raised a new fundamental question: whether there exists a unique aging program that governs the cellular dynamics of organ aging in large solid organs such as the skin.

[0003] Cell competition has been reported mainly in Drosophila to eliminate inappropriate mutant cells that arise in epithelial tissues during embryonic development and cancer development. Stem cells are known to participate in "neutral stem cell competition," characterized by stochastic stem cell loss and replacement, in some epithelial tissues. However, it has not been clear whether this is a completely random cell fate selection or a random cell competition caused by differences between cells. In the epidermis, symmetric cell division (SCD) and asymmetric cell division (ACD) of epidermal stem cells (keratinocyte stem cells and epidermal basal cells) maintain homeostasis of the epidermis by balancing them. However, the specific involvement and role of cell competition in the maintenance of physiological homeostasis and aging of mammalian organs, and in the cell dynamics of stratified epithelia, have been unknown.

[0004] With age, human skin exhibits skin atrophy (thinning), fragility, dryness, pigmentation abnormalities, delayed skin wound healing, and irritability. In the process, the skin loses its epidermal and dermal thickness, epidermal ridges, epidermal keratinocytes, and functional dermal fibroblast reserves. The fragility of human aged skin is partially attributed to remodeling of the dermal-epidermal junction, especially hemidesmosome (HD) components (structures that connect epithelial cells to the extracellular matrix of the basement membrane). Indeed, destabilization of HD components such as type XVII collagen (COL17A1 / BP180 / BPAG2 / type 17 collagen) in human aged skin has been reported. Furthermore, deficiency of the COL17A1 gene causes benign generalized atrophic epidermolysis bullosa (GABEB), a rare form of junctional epidermolysis bullosa (JEB), characterized by relatively mild skin fragility, atrophy, skin pigmentation abnormalities (poikiloderma) and alopecia. Consistent with the phenotype, previous studies have shown that Col17a1 gene deficiency in mice causes hair follicle aging accompanied by loss of hair follicle cells (Non-Patent Document 2, Patent Document 1). However, the relationship between the expression level of COL17A1 in epidermal stem cells and the expression of aging traits typically seen in healthy human skin (skin atrophy, fragility, impaired barrier function, pigmentation abnormalities, hair loss, etc.) was unclear. Furthermore, it was not clear how COL17A1 expression in epidermal stem cells is involved in maintaining skin youth, aging, and regeneration (wound healing), or its role, or the correlation between COL17A1 expression level and the competitive self-renewal ability of stem cells, or whether stem cell competition by COL17A1 maintains the quality (youth) of stem cells. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication 2009-161509 [Patent Document 2] International publication brochure WO2017122668 [Non-patent literature]

[0006] [Non-Patent Document 1] Matsumura, H. et al. Hair follicle aging is driven by transepidermal elimination of stem cells via COL17A1 proteolysis. Science 351, aad4395 (2016). [Non-Patent Document 2] Tanimura et al., Cell Stem Cell Vol.8, February 2011, pp.177-187 Summary of the Invention [Problem to be solved by the invention]

[0007] One of the objects of the present invention is to provide a method and composition for preventing and improving skin ulcers, that is, for promoting skin wound healing. Another object of the present invention is to provide a method and composition for reducing or preventing skin disorders associated with cancer treatment. Another object of the present invention is to provide a method for inhibiting skin aging, a composition for use in inhibiting skin aging, and a composition for inhibiting aging of hair follicles, which are skin appendages. Another object of the present invention is to provide a method for enhancing the regenerative ability of epidermal stem cells, a composition for enhancing the regenerative ability of epidermal stem cells (which may also include hair follicle stem cells), and a composition and method for controlling stem cell competition.

[0008] In addition, another object of the present invention is to provide a method for screening substances that are effective in reducing or preventing skin damage associated with cancer treatment, promoting skin wound healing, inhibiting skin aging, controlling cell competition, and / or improving the regenerative ability of epidermal stem cells. [Means for solving the problem]

[0009] To clarify the aging mechanism of mammalian organs using mice with identical genetic and environmental backgrounds, we used tail skin from C57BL6 inbred mice, which exhibit age-related skin atrophy, fragility, pigmentation abnormalities, and impaired wound healing, as seen in aged human skin. We found that epidermal cell competition dynamics linked to COL17A1-mediated epidermal stem cell proliferation maintains skin homeostasis, thereby controlling aging of skin organs. We also performed in vivo cell fate tracing analysis of epidermal stem cells in aging mice and found that COL17A1 in the epidermis is involved in the maintenance of skin homeostasis. high and COL17A1 low The heterogeneous emergence of clones promotes their mutual cell competition through COL17A1-mediated SCD, and COL17A1 low COL17A1 linked to clonal exclusion high We have demonstrated that COL17A1 promotes the proliferation of epidermal stem cell clones. Furthermore, we have shown that epidermal stem cell competition dynamics mediates the epidermal aging process, which is accompanied by a decline in skin regeneration capacity and a decrease in epidermal melanocytes and dermal mesenchymal cells, thereby regulating the aging of the skin organ itself. We also successfully prevented skin aging and improved wound healing by forcibly maintaining the expression of COL17A1 in epidermal stem cells and preserving the competitive self-renewal ability of epidermal stem cells, demonstrating that COL17A1 regulates the aging of the skin organ through the competition of epidermal stem cells and the maintenance of heterogeneous cells.

[0010] Cell competition has been implicated in the elimination of inappropriate cells, but its role in mammalian organ aging has been largely unknown until now. We show that epidermal stem cells sense their cytocompatibility / stress via the stability of HD component collagen XVII (COL17A1) to promote cell competition for skin homeostasis and aging. In vivo cell fate tracking, clonal analysis, and in vitro three-dimensional modeling reveal that COL17A1 high Epidermal stem cells clonally expand through COL17A1-dependent symmetric cell division, thereby removing COL17A1 from the skin to maintain homeostasis.low / - It has been found that COL17A1 expression in epidermis is essential for the elimination of exhausted cells. Their dominant expansion by competition ultimately minimizes their own COL17A1 levels, reducing not only their self-renewal capacity but also the regenerative capacity of adjacent epidermal melanocytes and dermal fibroblasts, leading to skin atrophy, pigmentation abnormalities and poor wound healing. Furthermore, forced maintenance of COL17A1 expression in the epidermis rescues the phenotype of skin aging. These findings open new avenues for therapeutic intervention. The present invention is based on these findings and specifically relates to the following:

[0011] [Aspect 1] A composition for use in promoting skin wound healing or preventing or improving skin ulcers or bedsores, characterized in that it contains as an active ingredient a substance selected from the group consisting of substances that induce or maintain the expression of COL17A1 in cells, substances that suppress the degradation of COL17A1 in cells, substances that maintain the self-renewal ability of epidermal stem cells, substances that suppress genomic stress or oxidative stress in cells, and substances that suppress DNA damage in cells. [Aspect 2] A composition for use in inhibiting skin aging, characterized in that it contains as an active ingredient a substance selected from the group consisting of substances that induce or maintain the expression of COL17A1 in cells, substances that inhibit the degradation of COL17A1 in cells, substances that maintain the self-renewal ability of epidermal stem cells, substances that inhibit genomic stress or oxidative stress in cells, and substances that inhibit DNA damage in cells. [Aspect 3] A composition for enhancing the regenerative ability of epidermal stem cells, characterized in that it contains as an active ingredient a substance selected from the group consisting of a substance that induces or maintains the expression of COL17A1 in cells, a substance that inhibits the degradation of COL17A1 in cells, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that inhibits genomic stress or oxidative stress in cells, and a substance that inhibits DNA damage in cells. [Aspect 4] A composition for use in preventing or improving skin damage caused by anticancer drugs, characterized in that it contains as an active ingredient a substance selected from the group consisting of substances that induce or maintain the expression of COL17A1 in cells, substances that suppress the degradation of COL17A1 in cells, substances that maintain the self-renewal ability of epidermal stem cells, substances that suppress genomic stress or oxidative stress in cells, and substances that suppress DNA damage in cells. [Aspect 5] A composition for use in anti-wrinkle treatment, characterized in that it contains as an active ingredient a substance selected from the group consisting of substances that induce or maintain the expression of COL17A1 in cells, substances that suppress the degradation of COL17A1 in cells, substances that maintain the self-renewal ability of epidermal stem cells, substances that suppress genomic stress or oxidative stress in cells, and substances that suppress DNA damage in cells. [Aspect 6] The composition according to embodiment 5, wherein the anti-wrinkle effect is achieved by at least one selected from the group consisting of improvement in skin elasticity, improvement in stratum corneum function, improvement in skin moisturizing ability, and improvement in skin barrier function. [Aspect 7] A composition for use in anti-spot treatment, characterized in that it contains as an active ingredient a substance selected from the group consisting of substances that induce or maintain the expression of COL17A1 in cells, substances that suppress the degradation of COL17A1 in cells, substances that maintain the self-renewal ability of epidermal stem cells, substances that suppress genomic stress or oxidative stress in cells, and substances that suppress DNA damage in cells. [Aspect 8] A composition for use in improving rough skin, characterized in that it contains as an active ingredient a substance selected from the group consisting of substances that induce or maintain the expression of COL17A1 in cells, substances that suppress the degradation of COL17A1 in cells, substances that maintain the self-renewal ability of epidermal stem cells, substances that suppress genomic stress or oxidative stress in cells, and substances that suppress DNA damage in cells. [Aspect 9] The composition according to any one of embodiments 1 to 8, wherein the substance that induces or maintains the expression of COL17A1 in cells further maintains the self-renewal ability of epidermal stem cells. [Aspect 10] The composition according to any one of embodiments 1 to 9, wherein the substance that induces or maintains expression of COL17A1 in a cell is selected from the group consisting of an NADPH oxidase inhibitor, a COX inhibitor, an iNOS inhibitor, a ROCK inhibitor, and an estrogenic substance. [Aspect 11] The composition of embodiment 10, wherein the NADPH oxidase inhibitor is selected from the group consisting of apocynin, ebselen, diphenyleneiodonium (DPI), GKT137831, AEBSF, GK-136901, ML171, Coenzyme Q10 (CoQ10), VAS2870, and VAS3947. [Aspect 12] 11. The composition of embodiment 10, wherein the COX inhibitor is selected from the group consisting of celecoxib, deracoxib, tolfenamic acid, nifluric acid, FR122047, LM-1685, SC-791, BTB02472, nimesulide, SPB04674, curcumin, diclofenac, 4'-hydroxydiclofenac, DuP-697, ebselen, ETYA, flurbiprofen, ibuprofen, indomethacin, meloxicam, NPPB, NS-398, pterostilbene, resveratrol, SC-560, SKF-86002, TXA (tranexamic acid), TXC (tranexamic acid cetyl hydrochloride), ginseng extract, and sulindac sulfide. [Aspect 13] 11. The composition of embodiment 10, wherein the iNOS inhibitor is selected from the group consisting of 1400W, L-NIL, aminoguanidine, BYK190123, S-ethylisothiourea, S-methylisothiourea, S-aminoethylisothiourea, 2-iminopiperidine, butylamine, ONO-1714 ((1S,5S,6R,7R)-7-chloro-3-imino-5-methyl-2-azabicyclo[4.1.0]heptane hydrochloride), AMT hydrochloride (2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine hydrochloride), AR-C102222, L-NG-nitroarginine, L-NG-monomethylarginine, L-nitroarginine methyl ester, L-NIO, dexamethasone, estrogen, astaxanthin, and apigenin. [Aspect 14] The composition of embodiment 10, wherein the ROCK inhibitor is selected from the group consisting of Y-27632, Ripasudil (K-115), Thiazovivin, Fasudil (HA-1077), GSK429286A, RKI-1447, GSK269962, Netarsudil (AR-13324), Y-39983, ZINC00881524, KD025, Hydroxyfasudil (HA-1100), GSK180736A and AT13148. [Aspect 15] The composition of embodiment 10, wherein the estrogenic substance is selected from the group consisting of estrogen, ethinyl estradiol, biochanin A, soybean extract, isoflavones, Belamcanda chinensis extract, and Pueraria mirifica root extract. [Aspect 16] The composition according to any one of aspects 1 to 9, wherein the substance that induces or maintains expression of COL17A1 in a cell is selected from the group consisting of apocynin, ebselen, celecoxib, apigenin, Y-27632, ripasudil, 1400W, ethinyl estradiol, biochanin A, necrostatin 1, rafuma extract, mulberry extract, gymnema extract, tea extract, loquat leaf extract, milk thistle extract, black turmeric extract, psyllium oleracea seed extract, coffee seed extract, Somei-Yodoshino leaf extract, Melia azadirachta leaf extract, Mandarin orange peel extract, lavender flower extract, Sophora angustifolia root extract, rice bran extract, Rosa canina fruit extract, Hamemaris leaf extract, Eucalyptus leaf extract, Crataegus crenata extract, and carrot extract. [Aspect 17] The composition according to any one of embodiments 1 to 9, wherein the substance that inhibits degradation of COL17A1 in cells is selected from the group consisting of a neutrophil elastase (ELANE) inhibitor and a matrix metalloproteinase (MMP) inhibitor. [Aspect 18] The ELANE inhibitor is sivelestat sodium hydrate (Monosodium N-{2-[4-(2,2-dimethylpropanoyloxy)-phenylsulfonylamino]benzoyl}aminoacetate tetrahydrate), ONO-6818 (2-(5-Amino-6-oxo-2-phenylhydropyrimidinyl)-N-[2-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-1-(methylethyl)-2-oxoethyl]acetamide), alpha 1 antitrypsin (alpha 1-AT), Depelestat, Nilone (Sodium trifluoride isopropyl oxopropyl aminocarbonyl pyrrolidine carbonyl methylpropyl aminocarbonyl benzoyl aminoacetate), perilla leaf fermentation product, parsley fermentation product, bell pepper fermentation product, an antibody against ELANE, an siRNA against a gene encoding ELANE, and an antisense oligonucleotide against a gene encoding ELANE. [Aspect 19] The composition of embodiment 17, wherein the MMP inhibitor is selected from the group consisting of Marimastat, Batimastat, PD166793, Ro32-3555, WAY170523, UK370106, TIMP1, TIMP2, TIMP3, and TIMP4. [Aspect 20] The composition according to any one of aspects 1 to 9, wherein the genomic stress is any one of DNA damaging stress caused by ultraviolet light, radiation or an anticancer drug, and replication stress associated with cell division. [Aspect 21] The composition according to embodiment 2, wherein the skin aging is at least one selected from the group consisting of the following (a) to (f): (a) Thinning, weakening, atrophy, or loss of firmness of the epidermis, dermis, and / or fat tissue (b) Decreased epidermal barrier function or dry skin (c) Reduction or immaturity of hemidesmosomes in the basement membrane (d) Loss of fibroblasts in the upper dermis or dry skin resulting in crinkled or fine wrinkles (e) Wrinkles due to loss of collagen in the dermis (f) Age spots, age spots, or depigmentation [Aspect 22] 3. The composition according to embodiment 2 for use in inhibiting wrinkles. [Aspect 23] The composition according to any one of embodiments 1 to 22, which is a pharmaceutical composition. [Aspect 24] The composition according to any one of the preceding aspects, further comprising a pharma- ceutically acceptable excipient. [Aspect 25] The composition according to any one of embodiments 1 to 24, which is an application agent. [Aspect 26] The composition according to any one of embodiments 1 to 22, which is a cosmetic composition. [Aspect 27] 27. The cosmetic composition according to claim 26, comprising 0.01 to 15% by weight of the active ingredient. [Aspect 28] 28. The cosmetic composition according to embodiment 26 or 27, which is a composition for use in skin care. [Aspect 29] 29. The cosmetic composition of any one of embodiments 26-28, further comprising at least one of a skin anti-aging agent, a skin toning agent, an anti-inflammatory agent, and a sunscreen agent. [Aspect 30] A method for screening a substance effective in promoting skin wound healing or preventing or ameliorating skin ulcers, inhibiting skin aging, controlling cell competition, improving the regenerative ability of epidermal stem cells, preventing or ameliorating skin disorders caused by anticancer drugs, preventing wrinkles, and / or improving rough skin, comprising: i) contacting a cell with a test substance in vitro; ii) measuring the expression of COL17A1 in the cells; and iii) determining whether the test substance increases the expression of COL17A1. A method comprising: [Aspect 31] A method for evaluating skin aging using the expression of COL17A1 in cells as an indicator. [Aspect 32] A kit for use in a method for evaluating skin aging, comprising an antibody against COL17A1, or a probe for a gene encoding COL17A1, or a primer for amplifying the gene. [Aspect 33] A method for evaluating epidermal stem cells using the expression of COL17A1 in the cells as an index. [Aspect 34] A kit for use in a method for evaluating epidermal stem cells, comprising an antibody against COL17A1, or a probe for a gene encoding COL17A1, or a primer for amplifying said gene. [Aspect 35] A method for selecting epidermal stem cells using the expression of COL17A1 in the cells as an indicator. [Aspect 36] A kit for use in a method for selecting epidermal stem cells, comprising an antibody against COL17A1, or a probe for a gene encoding COL17A1, or a primer for amplifying the gene. [Aspect 37] A method for expanding epidermal stem cells and / or epidermal cells, comprising the steps of measuring the expression level of COL17A1 using an antibody against COL17A1, or a probe for the gene encoding COL17A1, or a primer for amplifying the gene, and selecting epidermal stem cells having high expression levels of COL17A1. [Aspect 38] A kit for use in a method for amplifying epidermal stem cells and / or epidermal cells, comprising an antibody against COL17A1, or a probe for a gene encoding COL17A1, or a primer for amplifying the gene. [Aspect 39] A functional food or beauty supplement characterized by containing as an active ingredient a substance selected from the group consisting of substances that induce or maintain the expression of COL17A1 in cells, substances that inhibit the degradation of COL17A1 in cells, substances that maintain the self-renewal ability of epidermal stem cells, substances that inhibit genomic stress or oxidative stress in cells, and substances that inhibit DNA damage in cells. [Aspect 40] The functional food or cosmetic supplement according to embodiment 39, wherein the substance that induces or maintains the expression of COL17A1 in cells is selected from the group consisting of NADPH oxidase inhibitors, COX inhibitors, iNOS inhibitors, ROCK inhibitors and estrogenic substances. [Aspect 41] The functional food or beauty supplement according to aspect 39 or 40, wherein the substance that induces or maintains expression of COL17A1 in a cell is selected from the group consisting of apocynin, ebselen, celecoxib, apigenin, Y-27632, ripasudil, 1400W, ethinyl estradiol, biochanin A, necrostatin 1, rafuma extract, mulberry extract, gymnema extract, tea extract, loquat leaf extract, milk thistle extract, black turmeric extract, psyllium oleracea seed extract, coffee seed extract, Somei-Yodoshino leaf extract, Melia azadirachta leaf extract, Mandarin orange peel extract, lavender flower extract, Sophora angustifolia root extract, rice bran extract, Rosa canina fruit extract, Hamemaris leaf extract, Eucalyptus leaf extract, Crataegus crenata extract, and carrot extract. [Aspect 42] 42. The functional food or beauty supplement according to any one of aspects 39 to 41, which is for oral intake. [Aspect 43] 43. The functional food or beauty supplement according to any one of embodiments 39 to 42, in the form of a tablet, powder, semi-solid, jelly or liquid. [Aspect 44] 44. The functional food or beauty supplement of any one of embodiments 39-43, further comprising at least one of a skin anti-aging agent, a vitamin, collagen, and a mineral. [Aspect 45] A cell composition comprising an isolated cell that expresses COL17A1. [Aspect 46] The cell composition of embodiment 45, wherein the cells are epidermal keratinocytes or mucosal epithelial keratinocytes. [Aspect 47] 47. The cell composition of embodiment 45 or 46 for use in transplantation. [Aspect 48] 48. The cell composition of any one of embodiments 45 to 47 for use in treating a skin disease or injury. [Aspect 49] At least 1×10 3 The cell composition of any one of embodiments 45 to 48, comprising cells. [Aspect 50] The cell composition of any one of embodiments 45-49, wherein at least 50% of the cells express COL17A1. [Aspect 51] The cell composition according to any one of embodiments 45 to 50, wherein the cells are cells derived from stem cells. [Aspect 52] The cell composition of any one of embodiments 45 to 51, wherein the cells are frozen. [Aspect 53] The cell composition of any one of embodiments 45 to 52, wherein the cells are contained in a single container. [Aspect 54] The cell composition according to any one of embodiments 45 to 53, wherein the cells are cells that have been treated with a substance that induces or maintains expression of COL17A1 in the cells. [Aspect 55] The cell composition of embodiment 53, wherein the substance that induces or maintains expression of COL17A1 in the cells is selected from the group consisting of an NADPH oxidase inhibitor, a COX inhibitor, an iNOS inhibitor, a ROCK inhibitor, and an estrogenic substance. [Aspect 56] 56. The cell composition of embodiment 54 or 55, wherein the substance that induces or maintains expression of COL17A1 in the cells is selected from the group consisting of apocynin, ebselen, celecoxib, apigenin, Y-27632, ripasudil, 1400W, ethinyl estradiol, biochanin A, necrostatin 1, rafuma extract, mulberry extract, gymnema extract, tea extract, loquat leaf extract, milk thistle extract, black turmeric extract, psyllium oleracea seed extract, coffee seed extract, Somei-Yodoshino leaf extract, Melia azadirachta leaf extract, Mandarin orange peel extract, lavender flower extract, Sophora angustifolia root extract, rice bran extract, Rosa canina fruit extract, Hamemaris leaf extract, Eucalyptus leaf extract, Crataegus crenata extract, and carrot extract. [Aspect 57] A cell sheet comprising the cell composition according to any one of embodiments 45 to 56. [Aspect 58] A composition for use in controlling cell competition, characterized in that it contains as an active ingredient a substance selected from the group consisting of a substance that induces or maintains the expression of COL17A1 in a cell, a substance that suppresses the degradation of COL17A1 in a cell, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that suppresses genomic stress or oxidative stress in a cell, and a substance that suppresses DNA damage in a cell. [Aspect 59] A method for evaluating cell competition in keratinocytes by culturing keratinocytes in three dimensions. [Aspect 60] A kit for use in a method for evaluating cell competition in keratinocytes, comprising keratinocytes and a three-dimensional culture device. [Brief description of the drawings]

[0012] [Figure 1] Graph showing quantification of the fluorescence intensity of HD and basement membrane components, COL17A1, ITGA6, PLECTIN, ITGB4, COL7A1 and ITGB1, in the tails of young (7-8 weeks old, n=50) and old (22-25 months old, n=50) mice. Expression of COL17A1 and PLECTIN is significantly decreased in aged skin. [Diagram 2] Graph quantifying the number of HDs in the basement membrane of tail basal cells from young (8 weeks old, n=3) and old (22 months old, n=3) mice. Physiological aging significantly reduces the number of HDs in tail basal cells. [Diagram 3] Graphs quantifying the area (µm2) (left) and number (right) of monochromatic clones in whole-mount images of aging tail epidermis (4.5 months old, n=5 images; 15 months old, n=4 images; 24 months old, n=7 images). [Figure 4] Immunostaining of COL17A1 in multicolor-labeled tail basal keratinocytes from young (11 weeks old) and old (28 months old) tamoxifen (TAM)-treated Epi-Confetti mice. [Diagram 5] Graph quantifying the distribution between COL17A1 intensity and keratinocyte clone size in multiple regions (11-week-old, n = 13 clones; 28-month-old, n = 12 clones) in Figure 4 . [Figure 6] Graphs quantifying floating clones in control (Cont) mice (D2, n = 4; D28, n = 5) and Col17a1 cKO mice (D2, n = 3; D28, n = 5) on days 2 (D2) and 28 (D28) after TAM administration. [Figure 7] Graph showing colony formation in tail epidermal keratinocytes from young (7 weeks old, n=3) and old (25 months old, n=3) mice. The number and size of colonies are significantly decreased with age. [Figure 8]Graph analyzing colony formation in tail epidermal keratinocytes from wild-type (8 weeks old, n=3) and hCOL17A1 tg (8 weeks old, n=3) mice. Expression of hCOL17A1 tg significantly increased the number and size of colonies in primary tail epidermal keratinocytes. [Figure 9] Graph showing age-related changes in melanocytes. Age-related loss of melanocytes in the epidermis leads to skin pigmentation abnormalities (a mixture of deposition and loss) (left and center), but forced maintenance of COL17A1 maintains melanocytes and suppresses pigmentation abnormalities (right). [Figure 10] Graph showing the number of PDGFRa+ cells in wild-type and hCOL17A1tg mice, demonstrating the age-related decrease in PDGFRa+ cells just below the basement membrane (BM) (left) and the suppression of this decrease by forced maintenance of COL17A1 (right). [Figure 11] Graphs quantifying unrepaired wound area at days 0 (D0), 14 (D14) and 21 (D21). Physiological aging (left) or Col17a1 or Itga6 deficiency (right) significantly slows wound repair. [Figure 12] Graphs quantifying unrepaired wound area on days 0 (D0), 14 (D14), and 21 (D21). Expression of hCOL17A1 (left), and administration of apocynin or Y-27632 (right) significantly promoted wound repair. [Figure 13] Graph showing the results of colony formation assay. The number of colonies (n = 3 wells) was significantly increased by Y-27632 treatment compared to the control (Cont) (left). In addition, the number of colonies with a diameter of more than 2 mm (n = 3 wells) was significantly increased by Y-27632 or apocynin treatment (right). [Figure 14] Graph showing the results of a colony formation assay: The average size of the top 5 colonies (n=3 wells) was significantly increased by treatment with Y-27632 or apocynin compared to the control (Cont). [Figure 15]Graph of HCS analysis of COL17A1 expression (corresponding to Step 1 of screening for competitive self-renewal promoters mediated by COL17A1). Treatment with apocynin or ebselen in HCS significantly increased the amount of COL17A1 compared to the control. [Figure 16] Photographs of colony formation assay (corresponding to Step 2 of screening for competitive self-renewal promoters mediated by COL17A1 expression) and a graph analyzing the results. Treatment with apocynin or ebselen significantly increased the number and area of ​​colonies per well compared to the control. [Figure 17] Reduction in COL17A1 expression due to anticancer drugs and various stresses. Top: Immunostaining images examining COL17A1 expression in the skin of C57BL / 6 mice three months after continuous administration of hydroxyurea (HU). Center left: Western blotting image examining the amount of COL17A1 24 hours after HaCaT cells were exposed to UV light. Center: Western blotting image examining the amount of COL17A1 72 hours after HaCaT cells were exposed to radiation. Center right: Western blotting image examining the amount of COL17A1 24 hours after HaCaT cells were treated with hydrogen peroxide. Bottom: Western blotting image examining the amount of COL17A1 when HaCaT cells were treated with an EGF receptor inhibitor (PD168393). [Figure 18] Graph showing the ameliorative effect of COL17A1 overexpression on skin damage caused by anticancer drugs. [Figure 19] Graphs showing the effect of a competitive expander of epidermal stem cells on improving skin disorders caused by the administration of anticancer drugs. Left: Graph showing TEWL on the 12th day after continuous administration of an EGF receptor inhibitor (erlotinib) to 7-week-old C57BL / 6 mice. Right: Graph showing TEWL on the 7th day after continuous administration of an EGF receptor inhibitor (erlotinib) to 6-month-old C57BL6 mice. [Figure 20] A diagram showing the preventive and therapeutic effects of apocynin in a model of skin disorders (dry eczema and alopecia) induced in the upper back neck skin of aged mice. [Figure 21]Bar graphs showing the effect of substances that promote the cell competitiveness of epidermal stem cells on maintaining stratum corneum moisture content (top) and suppressing the increase in TEWL (bottom) in response to UV irradiation. [Figure 22] Bar graph (top) showing the inhibitory effect of a competitive amplifier of epidermal stem cells on the increase in TEWL caused by UV irradiation, and bar graph (bottom) showing the improving effect on reduced skin elasticity. [Figure 23] Graph (top) showing the effect of improving skin elasticity through long-term continuous application of a competitive amplifier of epidermal stem cells and a comparative agent. Bar graph (bottom) showing TEWL when a competitive amplifier of epidermal stem cells and a comparative agent are applied long-term. Retinol, known as an agent for improving wrinkles, significantly increases TEWL with long-term application. On the other hand, the TEWL value of the substance that promotes the cell competitive ability of epidermal stem cells is not significantly different from the control, suggesting that it has the effect of improving skin elasticity while maintaining the skin's barrier function. [Figure 24] High-resolution photographs and colored images of wrinkles (top) showing the effect of a competitive expander and a comparative agent on improving wrinkles caused by UV exposure. Also, bar graphs showing total wrinkle volume (mm3) and average total wrinkle depth (μm). [Diagram 25] A bar graph showing the TEWL when a competitive amplifier of epidermal stem cells was applied for a long period of time. Retinol, known as an anti-wrinkle agent, significantly increases TEWL with long-term application. On the other hand, the TEWL value of the substance that promotes the cell competitive ability of epidermal stem cells was not significantly different from the control, suggesting that it has the effect of improving wrinkles while maintaining the skin's barrier function. [Figure 26] 1 is a bar graph showing the effect of a substance that promotes the cell competitive ability of epidermal stem cells and a comparative agent in improving the loss of skin flexibility caused by ultraviolet radiation. [Figure 27] High-resolution photographs and colored images of skin texture showing the effect of a competitive amplifier of epidermal stem cells (apocynin) on improving rough skin induced by SDS. The graph shows the "Ra (arithmetic mean roughness)" and "Rz (ten-point mean roughness)", which are indices of roughness of the skin surface. [Figure 28]Bar graph showing the effect of apocynin, a substance that promotes the cell competitiveness of epidermal stem cells, in suppressing the increase in TEWL induced by SDS. [Figure 29] High-resolution photographs and colored images of skin texture showing the effect of a competitive expander of epidermal stem cells (ebselen) on improving rough skin induced by SDS. The graph shows "Ra (arithmetic mean roughness)" and "Rz (ten-point mean roughness)", which are indices of roughness of the skin surface. [Diagram 30] Bar graph showing the effect of a substance (ebselen) that promotes the cell competitiveness of epidermal stem cells in suppressing the increase in TEWL induced by SDS. [Diagram 31] Photographs showing the effects of competitive amplifiers of epidermal stem cells (apocynin, ebselen, celecoxib) on primary irritation skin damage (rough skin) and pigmentation induced by SDS. SDS treatment caused severe redness and dryness, but these were significantly alleviated by application of a competitive amplifier of epidermal stem cells. 16 days after the start of SDS treatment, it can be seen that the area where apocynin was applied had healed, but pigmentation had occurred in the control area. [Diagram 32] High-resolution photographs and colored images of skin texture showing the effect of competitive amplifiers of epidermal stem cells (apocynin, ebselen, celecoxib) on improving human skin roughness induced by SDS. The graph shows the values ​​of "Ry (maximum height)" and "Rz (ten-point average roughness)," which are indices of skin surface roughness, subtracted from the control on the measurement day, as "ΔRz" and "ΔRy." [Diagram 33] A graph showing the effect of competitive expanders of epidermal stem cells (apocynin, ebselen, and celecoxib) in suppressing the increase in TEWL associated with SDS-induced human rough skin. The error bars on the graph indicate standard deviation (SD). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] As described above, the present inventors have found that COL17A1 (type XVII collagen) is involved in skin aging and regeneration, and have identified a composition that is useful for promoting skin wound healing, inhibiting skin aging, controlling cell competition, and improving the regenerative ability of epidermal stem cells. The present invention will be described in detail below.

[0014] skin wound healing Skin wounds occur for various reasons, such as trauma, cuts, burns, poor circulation, ulcers due to bedsores, diabetes, etc., and temporarily impair the normal function and structure of the skin. If the body is unable to heal these wounds, they will become chronic and eventually suppurate. Skin wounds are classified as acute and chronic. Acute skin wounds are wounds in which the wound healing mechanism works normally, such as fresh trauma and surgical wounds, while chronic skin wounds are wounds in which the normal wound healing mechanism does not work due to some cause. The causes of delayed healing of chronic skin wounds can be broadly divided into two categories: systemic factors such as underlying diseases, and local factors. 1% of patients develop chronic skin wounds, but half of these wounds do not heal completely.

[0015] Skin is the largest organ in the human body and acts as a natural barrier against the outside world, protecting the tissues inside from wear and infection. Skin is generally divided into three layers: the epidermis, dermis, and subcutaneous tissue. The epidermis is the outermost layer and contains keratinocytes (keratinized cells) as well as melanocytes (pigment cells). Keratinocytes form the epidermis, which is arranged in multiple layers and covers the outer surface of the body. Melanocytes are distributed in the basal layer of the epidermis and provide melanin pigment to the surrounding keratinocytes. The distribution of pigment cells as well as the amount of melanin pigment and type of melanin determine the color of the skin.

[0016] The epidermis is further divided into four layers, from deepest to deepest: the basal layer (basal cell layer), the spinous layer (spinous cell layer), the granular layer (granular cell layer), and the stratum corneum (keratinocyte layer). The basal layer consists of a single layer of basal cells that contain keratinocyte stem cells. The basal layer has desmosomes, gap junctions, and hemidesmosomes as structures that connect it to adjacent cells and the basement membrane beneath the basal cells. The spinous layer consists of 5 to 10 layers. In this layer, the cells appear to be connected to each other by spines, so they are called spinous cells. The granular layer consists of 2 to 3 layers. It is called this because it contains keratohyalin granules that are basophilic and rich in profilaggrin. The stratum corneum, also called the stratum corneum, consists of about 10 layers. The dead keratinocytes that have been denucleated become membranous and are stratified like a layer of fallen leaves. The stratum corneum is extremely thick on the palms and soles of the feet, with a stratum lucidum just below it. The cytoplasm of the keratinocytes is filled with aggregated keratin fibers. Furthermore, hair follicles and sweat glands are connected to the epidermis, and act as skin appendages to grow hair and excrete sweat.

[0017] Epidermal stem cells present in the basal layer of the epidermis are the driving force behind epidermal regeneration, dividing horizontally against the basement membrane (equal division) or vertically (unequal division) in a well-balanced manner. They maintain the structure of the epidermis by self-replicating and supplying differentiated epidermal keratinocytes to the skin surface. Keratinocytes make up the majority of the cells in the epidermis. Almost all of the cells found in the stratum corneum on the skin surface are dead cells, and when these dead cells peel off, new keratinocytes rise from the lower layers to replace them. This process of rising from the lower layers is constantly repeated, and the entire skin surface is renewed with new ones in 15 to 30 days.

[0018] Beneath the epidermis lies a basement membrane, which separates it from the dermis layer below. The dermis layer is made up of fibroblasts, blood vessels, immune cells, skin appendages (hair follicles, sweat glands), and the extracellular matrix that supports these cells. Blood vessels nourish the skin, immune cells protect the skin, and fibroblasts produce collagen protein to give it strength and elastin protein to give it elasticity.

[0019] The deepest layer of skin is the subcutaneous tissue, a layer of fat beneath the dermis that provides cushioning for the underlying muscle and bone and acts as another barrier against infection.

[0020] Wounds form when the epidermal layer of the skin is destroyed by trauma, infection, anti-cancer drugs, age-related skin atrophy, barrier dysfunction, or other skin diseases, and the severity of wounds increases when the underlying layers of the skin become damaged. Three steps are required for a wound to heal normally: inflammation, proliferation, and remodeling. Inflammation lasts for about four days. The main thing that happens during this period is that a fibrin matrix is ​​created and a blood clot is formed to cover and protect the wound. During the proliferation phase, inflammatory signals attract many types of cells to the wound. These cells include fibroblasts and endothelial cells. Fibroblasts can produce collagen and transform into myofibroblasts to close the wound. Endothelial cells create new blood vessels around the wound. These cellular activities create a tissue called "granulation tissue" just below the blood clot. Granulation tissue is new tissue created as a repair and inflammatory response to tissue injury, and is composed of new blood vessels, connective tissue, fibroblasts, inflammatory cells, etc. Next, epidermal regeneration occurs from the epidermis surrounding the defect and from the dermal appendages, with keratinocytes migrating from the wound edge into the wound and from the base of the hair follicle. In deep skin defects without residual appendages, the wound surface is replaced by granulation tissue, and the epidermis extends from the periphery. Keratinocytes divide on the granulation tissue, uniting the epidermis. During the remodeling phase, this process continues, with epidermal recovery as epidermal keratinocytes migrate from the wound periphery or from the remaining appendages, and the interface between the epidermis and dermis is regenerated by the migration of epidermal keratinocytes from the wound periphery and the remaining appendages, producing matrix proteins of the newly formed basement membrane.

[0021] In cases of deep burns or cuts, where damage has been done to the deep dermis down to the fatty tissue, hair follicles and sweat glands may also be destroyed, leaving fewer keratinocytes available to repair the epidermis. As a result, full-thickness wounds take longer to heal and may require skin grafts, treatment with cultured skin such as epidermal sheets, or spray-applied keratinocyte grafts.

[0022] Composition for use in promoting skin wound healing or preventing or ameliorating skin ulcers or bedsores One embodiment of the present invention relates to a composition for use in promoting skin wound healing or preventing or improving skin ulcers or bedsores, characterized in that the composition contains as active ingredients a substance that induces or maintains the expression of COL17A1 in cells, a substance that suppresses the degradation of COL17A1 in cells, a substance that suppresses genomic stress or oxidative stress in cells, or a substance that suppresses DNA damage (DNA damage response or DNA damage itself) in cells (epidermal stem cells).

[0023] Substances that induce or maintain expression of COL17A1 in cells As described above, the present inventors have succeeded in preventing skin aging and improving or promoting wound healing, or preventing or improving skin ulcers or bedsores, by forcibly maintaining COL17A1 expression in epidermal stem cells, and have demonstrated that COL17A1 regulates aging of skin organs through competition between epidermal stem cells and maintenance of heterologous cells. The present inventors have also demonstrated that the matrix metalloproteinase (MMP) inhibitor marimastat stabilizes COL17A1 and blocks UV-induced down-repression of COL17A1. The present inventors have also identified a ROCK inhibitor (Y27632) and an NADPH oxidase inhibitor (apocynin) as chemicals that maintain or induce COL17A1 expression in keratinocytes in vitro and maintain and promote self-renewal ability in cultured keratinocytes.

[0024] Furthermore, the present inventors have shown that both drugs, Y27632 (ROCK inhibitor) and apocynin (NADPH oxidase inhibitor), significantly promote the wound repair process, as in transgenic mice overexpressing human COL17A1, both in vitro and in vivo. Thus, those skilled in the art will understand that a substance that induces or maintains the expression of COL17A1 in cells, or a substance that inhibits the degradation of COL17A1 in cells, can be used to promote or improve skin wound healing, or prevent or improve skin ulcers or bedsores. Furthermore, in the present invention, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that inhibits genomic stress or oxidative stress in cells, and a substance that inhibits DNA damage in cells can be used to promote or improve skin wound healing, or prevent or improve skin ulcers or bedsores.

[0025] COL17A1 (type XVII collagen / BP180 / BPAG2) is a transmembrane protein type cell adhesion molecule, and is a protein of hemidesmosomes (hemidesmosomes), which are one of the cell junctions in epithelial tissue. Examples of substances that induce or maintain the expression of COL17A1 in cells include, but are not limited to, ROCK inhibitors, NADPH oxidase inhibitors, iNOS inhibitors, and COX inhibitors. Substances that induce or maintain the expression of COL17A1 in cells also include DNA or RNA that codes for COL17A1. DNA or RNA that codes for COL17A1 may be introduced into cells using a suitable vector.

[0026] Examples of ROCK inhibitors include, but are not limited to, Y27632, thiazovivin, fasudil (HA-1077), GSK429286A, RKI-1447, GSK269962, netarsudil (AR-13324), Y-39983, ZINC00881524, KD025, ripasudil (K-115), hydroxyfasudil (HA-1100), GSK180736A, and AT13148. Examples of NADPH oxidase inhibitors include, but are not limited to, apocynin, ebselen, diphenyleneiodonium (DPI), GKT137831, AEBSF, GK-136901, ML171, Coenzyme Q10 (CoQ10), VAS2870, and VAS3947. Plant extracts containing apocynins, such as Rafuma extract, are also included.

[0027] Of the plant extracts used in the present invention, Rahmania extract (trade name: Venetron), mulberry extract (trade name: Mulberry leaf extract powder), Gymnema extract (trade name: Gymnema extract powder), tea extract (trade name: Tiacalon 90), loquat leaf extract (trade name: Loquat leaf extract powder), Milk thistle extract (trade name: Milk thistle extract powder), and black turmeric extract (trade name: Certmax) are available from Tokiwa Phytochemical Laboratory Co., Ltd. Plantago major seed extract (product name: Absolage), coffee seed extract (product name: Cafe Noage), Somei Yoshino cherry leaf extract (product name: Sakura Extract B), Melia azadirachta leaf extract (product name: Neem Leaf Liquid B), Mandarin orange peel extract (product name: Mandarin Culia), Lavender flower extract (product name: Ecofarm Lavender B), Sophora flavonoides root extract (product name: Falcorex Sophora flavonoides B), Rice bran extract (product name: Falcorex Rice Bran BK), Rosa canina extract (product name: Falcorex Rosa canina B), Hamemaris leaf extract (product name: Falcorex Hamemaris B), Eucalyptus leaf extract (product name: Falcorex Eucalyptus B), and Hawthorn extract (product name: Falcorex Hawthorn B) are listed in the Ichimaru Pharcos Co., Ltd. Product Guide and can be easily obtained.

[0028] As the iNOS inhibitor, either a selective iNOS inhibitor or a non-selective iNOS inhibitor can be used, but from the viewpoint of side effects, for example, a selective iNOS inhibitor is preferred. Examples of iNOS inhibitors include 1400W, L-NIL, aminoguanidine, BYK190123, S-ethylisothiourea, S-methylisothiourea, S-aminoethylisothiourea, 2-iminopiperidine, butylamine, ONO-1714 (fused piperidine derivative; (1S,5S,6R,7R)-7-chloro-3-imino-5-methyl-2-azabicyclo[4.1.0]heptanhydride These include, but are not limited to, arginine, arginine hydrochloride (2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine hydrochloride), AMT hydrochloride (2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine hydrochloride), AR-C102222, L-NG-nitroarginine, L-NG-monomethylarginine, L-nitroarginine methyl ester, L-NIO, dexamethasone, estrogen, astaxanthin, and the iNOS expression inhibitor apigenin.

[0029] Examples of COX inhibitors include, but are not limited to, celecoxib, deracoxib, tolfenamic acid, FR122047, LM-1685, SC-791, BTB02472, nimesulide, SPB04674, curcumin, diclofenac, 4'-hydroxydiclofenac, DuP-697, ebselen, ETYA, flurbiprofen, ibuprofen, indomethacin, meloxicam, niflunic acid, NPPB, NS-398, pterostilbene, resveratrol, SC-560, SKF-86002, TXA (tranexamic acid), TXC (tranexamic acid cetyl hydrochloride), carrot extract, astaxanthin, and sulindac sulfide. As the COX inhibitor, either a selective COX inhibitor or a non-selective COX inhibitor can be used, but from the viewpoint of side effects, for example, a selective COX-2 inhibitor is preferred.

[0030] Examples of estrogen-like substances include, but are not limited to, estrogen, ethinyl estradiol, biochanin A, soybean extract, isoflavone, Belamcanda chinensis extract, and Pueraria mirifica root extract.

[0031] Substances that inhibit the degradation of COL17A1 in cells Substances that suppress the degradation of COL17A1 in cells include, for example, neutrophil elastase (ELANE) inhibitors and MMP inhibitors. Examples of ELANE inhibitors include sivelestat sodium hydrate (Monosodium N-{2-[4-(2,2-dimethylpropanoyloxy)-phenylsulfonylamino]benzoyl}aminoacetate tetrahydrate; also known as Elaspol), ONO-6818 (2-(5-Amino-6-oxo-2-phenylhydropyrimidinyl)-N-[2-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-1-(methylethyl)-2-oxoethyl]acetamide), α1-antitrypsin (α1-AT; A1AT), and Depelestat (EPI Examples of elastase activity inhibitors include, but are not limited to, elastase activity inhibitors containing fermented perilla leaves (Japanese Patent Publication No. 2006-61091), fermented parsley (Japanese Patent Publication No. 2006-75085), and fermented bell peppers (Japanese Patent Publication No. 2006-76926).

[0032] The ELANE inhibitor may be an antibody against ELANE, an siRNA against a gene encoding ELANE, an antisense oligonucleotide against a gene encoding ELANE, etc. Examples of MMP inhibitors include, but are not limited to, Marimastat, Batimastat, PD166793, Ro32-3555, WAY170523, UK370106, TIMP1, TIMP2, TIMP3, TIMP4, etc.

[0033] Substances that maintain the self-renewal ability of epidermal stem cells The present inventors have discovered that the maintenance of epidermal stem cells is made possible by the expression and maintenance of COL17A1 and is based on the self-renewal ability of epidermal stem cells. Therefore, in the present invention, substances that maintain the self-renewal ability of epidermal stem cells include ROCK inhibitors, NADPH oxidase inhibitors, iNOS inhibitors, and COX inhibitors, as well as growth factors and plant extracts. For example, apocynin, ebselen, celecoxib, apigenin, Y-27632, ripasudil, 1400W, astaxanthin, ethinylestradiol, biochanin A, necrostatin 1, rafuma extract, mulberry extract, gymnema extract, tea extract, loquat leaf extract, milk thistle extract, black turmeric extract, psyllium seed extract, coffee seed extract, Somei-Yoshino leaf extract, Melia azadirachta leaf extract, mandarin orange peel extract, lavender flower extract, Sophora Angustifolia root extract, rice bran extract, Rosa canina fruit extract, Hamemaris leaf extract, eucalyptus leaf extract, hawthorn extract, carrot extract, etc.

[0034] These substances maintain or promote the self-renewal ability of epidermal stem cells. In vivo or 3D cultured epidermis, epidermal stem cells expressing sufficient amounts of COL17A1 compete with surrounding epidermal stem cells expressing low levels of COL17A1, competitively occupying the epidermis and expanding. Therefore, the self-renewal ability based on the expression of COL17A1 is called "competitive self-renewal ability." The self-renewal ability of epidermal stem cells can be exemplified by the competitive self-renewal ability of epidermal stem cells, and the above substances can also be called "competitive amplification agents for epidermal stem cells."

[0035] Substances that suppress genomic or oxidative stress in cells The present inventors also found that the expression of COL17A1 is decreased by various stresses. In the present invention, the stresses to be suppressed are genomic stress (genotoxic stress) and oxidative stress, and genomic stress includes genotoxic stress such as ultraviolet light, radiation stress, or DNA damage stress caused by anticancer drugs, or replication stress associated with cell division. The function of cells is reduced by stress that damages DNA or by blocking self-replication signals by molecular targeting drugs. Therefore, those skilled in the art will understand that skin wound healing can be promoted and improved by using a substance that suppresses genomic stress or oxidative stress in cells, or a substance that suppresses DNA damage in cells.

[0036] Substances that suppress DNA damage in cells Substances that suppress DNA damage include, for example, Sarcandra glabra extract, Saraca dives extract, Cudrania pubescens extract, Taxodium distichum extract, Ludwigia octovalis extract, Deutzianthus tonkinensis extract, Alchornea trewioides extract, Berchemia polyphylla extract, Glochidion puberum extract, Sassafrastum extract, and the like. Examples of usable herein include an extract of Gynostemma tzumu (see JP 2008-247854 A), cinchona extract, comfrey extract, coffee extract, pueraria root extract, burdock extract, coptis japonica extract, sophora flavescens extract, chlorella extract, lavender extract, evening primrose extract, rose extract, Gynostemma pentaphyllum extract, Enmeiso extract, Lamium extract, carrot extract, linden extract, Juglans japonica extract, lotus extract, tea extract, and okra extract (see WO2013 / 031003).

[0037] The DNA damage suppression ability of a substance that suppresses DNA damage can be evaluated using any method known to those skilled in the art, such as a comet assay or an assay that detects DNA damage foci induced by a DNA damage response. In addition, a substance that promotes DNA damage repair may be used as a substance that suppresses DNA damage. For example, a Gigartinatenella extract (see JP 2006-273761 A) or a Cinchona extract (see WO2013 / 031003 A) can be used as an agent that promotes DNA damage repair. The DNA damage repair ability of an agent that promotes DNA damage repair can be evaluated using any method known to those skilled in the art, such as a host-cell reactivation assay. Examples of substances that suppress genomic stress or oxidative stress in cells include, but are not limited to, antioxidants, ultraviolet absorbers, ultraviolet scattering agents, radiation protectors, and DNA damage repair promoters.

[0038] Composition for use in inhibiting skin aging One embodiment of the present invention relates to a composition for use in inhibiting skin aging, characterized in that the composition contains as active ingredients a substance that induces or maintains the expression of COL17A1 in cells, a substance that inhibits the degradation of COL17A1 in cells, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that inhibits genomic stress or oxidative stress in cells, and / or a substance that inhibits DNA damage in cells. One embodiment of the present invention may be a composition for use in inhibiting epidermal aging, in particular. Some embodiments of the present invention may be a composition for use in inhibiting aging of skin, in particular, including hair follicles. Also, some embodiments of the present invention may be a composition for use in inhibiting aging of skin, in particular, excluding hair follicles.

[0039] The composition for use in inhibiting skin aging contains the active ingredient described in the section "Composition for use in promoting skin wound healing, or preventing or improving skin ulcers or bedsores" above, and hereinafter in this specification the active ingredient of the composition can be applied in the same manner as above.

[0040] As we age, the skin's role of isolating an individual from the outside world and its regenerative ability decline. Skin function is maintained by epidermal stem cells present in the basal layer of the epidermis and their normal differentiation. The descendants of epidermal stem cells adhere and align perpendicularly to the skin surface, forming a layer of a certain thickness and undergoing normal keratinization, allowing the skin to retain its regenerative ability while retaining its strength as a barrier against the outside world and preventing erosion and ulcers. However, as we age or due to various stresses, the skin begins to show common characteristics that prevent it from fulfilling these roles, and these changes are collectively referred to as skin aging.

[0041] With aging, the skin's appearance becomes less fine and uneven, with fine wrinkles and fine lines. The barrier function of the epidermis is weakened and the skin becomes more prone to dryness, and the epidermis, dermis and / or fat tissue become thin or fragile, which makes the skin more susceptible to redness, eczema, sores, erosions and ulcers. In addition, the pigmentation of the skin becomes more pronounced. In general, wrinkles due to a decrease in collagen in the dermis, thinning (atrophy) of the epidermis, dermis and fat tissue, loss of firmness and fragility, as well as age spots, senile pigmentation spots and depigmentation spots are well-known characteristics of aged skin. The inventors have histologically found that common features of aged skin in mice and humans include epidermal atrophy, a decrease in pigment cells, a decrease or immaturity of hemidesmosome components in the basement membrane, particularly a decrease in COL17A1 and a decrease in hemidesmosomes, a disappearance of fibroblasts in the upper dermis, and crepe wrinkles and fine wrinkles due to dry skin, and that PDGFRα in the upper dermis is a factor in determining whether aging is caused by aging. + A decrease in mesenchymal cells was also observed.

[0042] It is known that COL17A1 deficiency makes the skin fragile, and the above findings may explain why aged skin is prone to wrinkles, erosions, and ulcers. Therefore, those skilled in the art will understand that skin aging can be suppressed using substances that induce or maintain COL17A1 expression in cells, substances that suppress COL17A1 degradation in cells, substances that suppress genomic or oxidative stress in cells, and / or substances that suppress DNA damage in cells.

[0043] One embodiment of the present invention is a composition for use against skin aging, comprising: (a) Thinning, weakening, atrophy, or loss of firmness of the epidermis, dermis, and / or fat tissue (b) Decreased epidermal barrier function or dry skin (c) Reduction or immaturity of hemidesmosomes in the basement membrane (d) Loss of fibroblasts in the upper dermis or dry skin resulting in crinkled or fine wrinkles (e) Wrinkles due to loss of collagen in the dermis (f) Age spots, age spots, or depigmentation One embodiment of the present invention is a composition specifically for use in inhibiting wrinkles.

[0044] One embodiment of the present invention relates to a composition for maintaining the regenerative ability or competitive self-renewal ability of epidermal stem cells, characterized in that the composition contains as active ingredients a substance that induces or maintains the expression of COL17A1 in cells, a substance that suppresses the degradation of COL17A1 in cells, a substance that suppresses genomic stress or oxidative stress in cells, and / or a substance that suppresses DNA damage in cells. In the context of this specification, skin aging is accompanied by one or more of the following: thinning of the epidermis, reduced barrier function, dryness, weakening of the basement membrane, wrinkle formation due to collagen reduction in the dermis, thinning, atrophy, loss of firmness, weakening, age spots, or senile pigmentation spots of the epidermis, dermis, and / or fat tissue. Epidermal stem cells are stem cells that exist in the basal layer of the epidermis and supply new epidermal keratinocytes, play an important role in skin metabolism, and have the function of keeping the skin surface in a healthy state. When epidermal stem cells become senescent due to aging, various genomic stresses, oxidative stress, or the rapid blockage of self-renewal signals by molecular targets, the intrinsic self-renewal ability of epidermal stem cells is reduced and they become more susceptible to being lost through terminal differentiation.

[0045] As described above, the present inventor has revealed that skin homeostasis is maintained by epidermal cell competition dynamics linked to COL17A1-mediated proliferation of epidermal stem cells, thereby controlling aging of skin organs. Epidermal stem cells with high expression of COL17A1 can be said to have high regenerative ability. Thus, those skilled in the art will understand that the regenerative ability of epidermal stem cells can be enhanced using a substance that induces or maintains the expression of COL17A1 in cells, a substance that inhibits the degradation of COL17A1 in cells, a substance that inhibits genomic or oxidative stress in cells, and / or a substance that inhibits DNA damage in cells. By enhancing the regenerative ability of epidermal stem cells and maintaining or improving the supply of new epidermal keratinocytes, skin aging is inhibited and the condition of the skin surface is maintained or improved.

[0046] Anti-wrinkle composition One embodiment of the present invention is a composition for use in anti-wrinkle, characterized in that it contains as an active ingredient a substance selected from the group consisting of a substance that induces or maintains the expression of COL17A1 in cells, a substance that suppresses the degradation of COL17A1 in cells, a substance that suppresses genomic stress or oxidative stress in cells, and a substance that suppresses DNA damage in cells.The anti-wrinkle effect includes at least one selected from the group consisting of improvement of skin elasticity, improvement of stratum corneum function, improvement of skin moisturizing ability, and improvement of skin barrier function.

[0047] Improves skin elasticity In the present invention, skin elasticity refers to mechanical properties such as flexibility (softness) and elasticity (the degree to which stretched skin returns to its original shape like a spring within a certain time). Skin elasticity is considered to be one index of skin age, and moist, elastic, spring-like skin is felt to be "soft" and "firm". It is believed that ultraviolet rays affect the decrease in skin elasticity, and the decrease in elasticity is noticeable in exposed areas such as the face that are exposed to ultraviolet rays. Skin elasticity is also called viscoelasticity, and can be quantitatively measured using Cutometer MPA580 (manufactured by Courage+Khazaka, Germany, Integral Co., Ltd.) by applying force to the skin to deform it and measuring the process until the skin returns to its original position. The Cutometer is a device commonly used in the fields of dermatology and cosmetic science, and its principle is to apply negative pressure to the skin using a probe with a hole of 2 mm in diameter to suck the skin, then release the negative pressure to measure the degree of return of the skin to measure viscoelasticity. The following values ​​were used in the analysis; it is generally believed that the higher the value, the better the elasticity, and this value decreases with age.

[0048] Ur / Uf value (R7): An index of elasticity expressed using the maximum elongation value (Uf or R0) and the height of elastic elongation at relaxation (Ur), which indicates the instantaneous return rate of the skin to maximum elongation. Ur / Ue (R5): An index of elasticity expressed using the height of elastic elongation at relaxation (Ur) and the height of elastic elongation when sucked into the probe (Ue). It indicates the instantaneous return rate of the skin to maximum elongation and is considered to be the net elasticity. Ua / Uf(R2): Elasticity index expressed using the maximum elongation value (Uf) and Ua, which indicates the instantaneous return rate of the skin to the maximum elongation, also called total elasticity. Ua is the difference between Uf and R1 (the height of the residual strain after the first measurement cycle).

[0049] Improves stratum corneum function Improvement of stratum corneum function means, when applied to the skin, improving the moisturizing ability of increasing or retaining moisture in the stratum corneum, and improving the barrier function of preventing the intrusion of foreign substances from the outside world, and a stratum corneum function improver is one that can exert either of these effects.

[0050] Improving skin moisturizing ability In the present invention, "moisture retention ability" refers to the retention of moisture in the stratum corneum, and the higher the moisture content of the stratum corneum, the better the moisture retention ability. An agent for improving skin moisture retention ability refers to an agent that can retain the moisture content of the stratum corneum.

[0051] Improves skin barrier function In addition, "barrier function" refers to the inhibition of transepidermal water loss, and the smaller the transepidermal water loss (TEWL), the better the barrier function. The skin barrier function prevents water from evaporating from the body and prevents foreign substances from entering the body, and its function is reduced by exposure to ultraviolet rays. In general, the moisture content of the stratum corneum, TEWL, and epidermal thickness are used as indicators of the skin barrier function, and are indicators of the degree of skin damage caused by ultraviolet rays. An agent that improves the skin barrier function is one that can prevent water evaporation or the entry of foreign substances from the body.

[0052] Improves rough skin Rough skin generally refers to a condition in which the smoothness of the skin surface is lost, causing dryness and other problems, as opposed to healthy skin that is well-textured and moisturized. In addition to superficial problems such as a rough feeling, redness, and unevenness of the skin, it may also be accompanied by itching. It is known that rough skin is closely related to a decrease in the skin barrier function. For this reason, a method is being used to quantitatively analyze and evaluate symptoms of rough skin by measuring TEWL, which is an index of skin barrier function. Furthermore, since rough skin causes roughness (unevenness) on the stratum corneum surface, rough skin can be quantitatively analyzed from values ​​such as arithmetic mean roughness (Ra), maximum height (Ry), and ten-point mean roughness (Rz), which are ISO standard surface roughness parameters for the skin. The skin surface is photographed using a three-dimensional high-resolution imaging device PRIMOS-CR, and the surface can be analyzed using dedicated software (Primos OMC3_22). Using a rough skin model that serves as the standard for chemical-induced skin damage in humans, the inventors discovered that a substance that maintains the self-renewal ability of epidermal stem cells improves rough skin by suppressing the increase in TEWL associated with artificially induced rough skin and improving the roughness of the skin surface.

[0053] Therefore, one embodiment of the present invention provides a composition for use in improving rough skin, characterized in that it contains as an active ingredient a substance selected from the group consisting of a substance that induces or maintains the expression of COL17A1 in cells, a substance that inhibits the degradation of COL17A1 in cells, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that inhibits genomic stress or oxidative stress in cells, and a substance that inhibits DNA damage in cells. Rough skin refers to a condition in which the skin loses its smoothness, moisture, and smooth appearance and feel, and is often accompanied by dryness, itching, redness, erosion, etc., and the composition of the present invention can improve the above symptoms or conditions.

[0054] Anti-stain composition A blemish is a deposit of melanin, a pigment produced within the skin. The most common is solar lentigo (senile lentigo), which occurs as a result of continued exposure to ultraviolet rays, but it also refers to a wide range of conditions in which the uniformity of pigment is disrupted, such as freckles, post-inflammatory hyperpigmentation, and liver spots. Therefore, "anti-blemish" refers to maintaining the uniformity of pigment. The present invention provides a composition for use in anti-spot treatment, characterized in that it contains as an active ingredient a substance selected from the group consisting of a substance that induces or maintains the expression of COL17A1 in cells, a substance that inhibits the degradation of COL17A1 in cells, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that inhibits genomic stress or oxidative stress in cells, and a substance that inhibits DNA damage in cells.

[0055] Preventive and remedial agents for skin disorders caused by anti-cancer drugs One embodiment of the present invention relates to a method and composition for preventing or ameliorating skin disorders associated with anti-cancer drug treatment.

[0056] Skin disorders (skin toxicity) frequently occur as a side effect of anticancer drug treatment. In addition to dry skin, hand-foot syndrome (palmar-plantar erythrodysesthesia syndrome), pigmentation, nail changes, and aging-like skin disorders are known. Metabolic antagonists, alkylating agents, and platinum preparations that have been used traditionally cause dry skin, atrophy, fragility, impaired barrier function, pigmentation abnormalities, and hair loss. In recent years, focusing on the differences between cancer cells and normal cells, molecular targeted drugs that selectively attack molecules specifically expressed in cancer cells have been developed and are being used in various cancers such as breast cancer and lung cancer, replacing conventional anticancer drugs.

[0057] In addition to skin disorders commonly seen with anticancer drugs (dry skin, atrophy, hand-foot syndrome, pigmentation abnormalities, hair loss, etc.), skin toxicity caused by molecular targeted drugs (various multi-tyrosine kinase inhibitors, EGF receptor inhibitors, etc.) frequently results in erythema-papular rash, acneiform rash, seborrheic dermatitis, hyperpigmentation of hair and nails, paronychia, dry and cracked skin, xerosis, itching, and strong stinging. Skin disorders have also been observed with new immunotherapy drugs (immune checkpoint inhibitors). Skin disorders caused by these molecular targeted drugs are not easily considered harmful, but are evaluated as a manifestation of pharmacological action on skin cells that express molecules common to cancer cells, or as an indicator of the effectiveness of anticancer drugs, and anticancer treatment is continued while continuing symptomatic treatment with moisturizers, etc. However, it causes great mental distress and burden for patients, and in many cases patients give up treatment on their own. In addition, there are many cases where the administration of anticancer drugs (chemotherapy and molecular targeted drugs) is discontinued due to severe skin disorders, but there are no appropriate preventive methods or local treatment methods.

[0058] Until now, it has been unknown how COL17A1 expression in epidermal stem cells is changed by many different anticancer drugs, how expression is reduced by molecular targeted drugs, how to suppress this, and the relationship with anticancer drugs. Although trial and error has been performed on symptomatic treatments such as applying moisturizers and steroids to the skin, no effective method has been developed to prevent or improve skin damage associated with cancer treatment.

[0059] In the present invention, it was revealed that not only anticancer drugs such as hydroxyurea but also molecular targeted drugs such as EGF receptor inhibitors reduce the expression of COL17A1 and the competitive self-renewal ability of stem cells. Therefore, the present invention provides a composition for use in preventing or improving skin damage caused by anticancer drugs, characterized in that it contains as an active ingredient a substance selected from the group consisting of a substance that induces or maintains the expression of COL17A1 in cells, a substance that inhibits the degradation of COL17A1 in cells, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that inhibits genomic stress or oxidative stress in cells, and a substance that inhibits DNA damage in cells.

[0060] In one embodiment, the agent for preventing and improving skin disorders according to the present invention may be characterized by containing, as an active ingredient, a substance that maintains the expression and self-replication ability of COL17A1 in cells. More specifically, the agent for preventing and improving skin disorders according to the present invention may contain, as a substance that induces or maintains the expression of COL17A1 in cells, a ROCK inhibitor, an NADPH oxidase inhibitor, an iNOS inhibitor, and / or a COX inhibitor. More specifically, the agent for preventing and improving skin disorders according to the present invention may contain, as substances that maintain the expression of COL17A1 in cells and maintain self-replication ability, apocynin, ebselen, celecoxib, apigenin, Y-27632, ripasudil, 1400W, astaxanthin, ethinyl estradiol, biochanin A, necrostatin 1, rafuma extract, mulberry extract, gymnema extract, tea extract, loquat leaf extract, milk thistle extract, black turmeric extract, psyllium asiatica seed extract, coffee seed extract, Somei-Yodoshino leaf extract, Melia azadirachta leaf extract, Mandarin orange peel extract, lavender flower extract, Sophora angustifolia root extract, rice bran extract, Rosa canina fruit extract, Hamemaris leaf extract, eucalyptus leaf extract, hawthorn extract, and carrot extract.

[0061] plant extract In the present invention, various plant extracts can be used for the applications mentioned above (below). - Promoting skin wound healing or preventing or improving skin ulcers ·Suppression of skin aging -Enhancing the regenerative ability of epidermal stem cells -Prevention or improvement of skin damage caused by anti-cancer drugs Anti-wrinkle (improvement of skin elasticity, improvement of stratum corneum function, improvement of skin moisturizing ability, and improvement of skin barrier function) Anti-stain Furthermore, in the present invention, the plant extract can also be used as a COL17A1 promoter and an agent for inhibiting hair loss and graying.

[0062] The raw materials of the plant extracts used in the present invention include Apocynum venetum, Mulberry (Morus alba), Gymnema (Gymnema sylvestre), Tea (Camellia sinensis), Milk thistle (Silybum marianum), Loquat leaf (Eriobotrya japonica), Black turmeric (Kaempferia parviflora), Ginseng (Panax ginseng), Ashwagandha (Indian ginseng), etc. The parts of these plants to be used are not limited as long as the above-mentioned effects can be obtained.

[0063] The method of extracting each plant extract is not particularly limited, and can be carried out according to a method well known to those skilled in the art. As the extraction solvent, water, alcoholic solvents, and organic solvents such as acetone, esters, polyhydric alcohols, and ethers can be used. Thus, for example, ethanol extracts, hot water extracts, 1,3-butylene glycol extracts, etc. of the above plant raw materials can be prepared. These solvents may be used alone or in combination. The amount of the extraction solvent, extraction temperature, extraction time, and extraction method are not limited as long as the composition exhibiting the above effects can be obtained.

[0064] The extract may be the filtrate obtained by filtering the extract, a concentrate obtained by concentrating the filtrate, a dried product obtained by drying the concentrate, or a crude or purified product thereof. Any method for concentrating or drying may be used. If necessary, an excipient such as dextrin may be added. Purification may be performed by any method known to those skilled in the art, such as synthetic adsorption resins, activated carbon, ion exchange resins, column chromatography, or recrystallization. The present composition may also take the form of a food composition, a pharmaceutical composition, or a cosmetic composition. In particular, in the case of a food composition, it may take the form of a functional food or health food.

[0065] Pharmaceutical Compositions The composition according to the present invention may be a pharmaceutical composition. One embodiment of the present invention relates to a pharmaceutical composition for use in promoting skin wound healing or preventing or improving skin ulcers or bedsores, characterized in that the pharmaceutical composition contains, as an active ingredient, a substance that induces or maintains the expression of COL17A1 in cells, a substance that suppresses the degradation of COL17A1 in cells, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that suppresses genomic stress or oxidative stress in cells, and / or a substance that suppresses DNA damage in cells.

[0066] The pharmaceutical composition of the present invention can be used for treating acute skin wounds or preventing or treating chronic skin wounds.In other words, one embodiment of the present invention relates to the use of a substance that induces or maintains the expression of COL17A1 in cells, a substance that inhibits the degradation of COL17A1 in cells, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that inhibits genomic or oxidative stress in cells, and / or a substance that inhibits DNA damage in cells in the manufacture of a medicament for use in promoting skin wound healing or preventing or improving skin ulcers or bedsores.

[0067] Furthermore, one embodiment of the present invention relates to a pharmaceutical composition for use in suppressing skin aging, characterized in that the pharmaceutical composition contains as active ingredients a substance that induces or maintains the expression of COL17A1 in cells, a substance that suppresses the degradation of COL17A1 in cells, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that suppresses genomic stress or oxidative stress in cells, and / or a substance that suppresses DNA damage in cells.

[0068] The pharmaceutical composition of the present invention can be used for preventing or treating skin symptoms of diseases accompanied by the progression of skin aging.In other words, one embodiment of the present invention relates to the use of a substance that induces or maintains the expression of COL17A1 in cells, a substance that inhibits the degradation of COL17A1 in cells, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that inhibits genomic stress or oxidative stress in cells, and / or a substance that inhibits DNA damage in cells in the manufacture of a medicine for use in inhibiting skin aging. Furthermore, one embodiment of the present invention relates to a pharmaceutical composition for enhancing the regenerative ability of epidermal stem cells, characterized in that it contains as active ingredients a substance that induces or maintains the expression of COL17A1 in cells, a substance that inhibits the degradation of COL17A1 in cells, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that inhibits genomic stress or oxidative stress in cells, and / or a substance that inhibits DNA damage in cells.

[0069] The pharmaceutical composition according to the present invention can be used for the prevention or treatment of skin symptoms of diseases accompanied by a decrease in the regenerative ability of epidermal stem cells. In other words, one embodiment of the present invention relates to the use of a substance that induces or maintains the expression of COL17A1 in cells, a substance that inhibits the degradation of COL17A1 in cells, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that inhibits genomic stress or oxidative stress in cells, and / or a substance that inhibits DNA damage in cells in the manufacture of a medicine for enhancing the regenerative ability (self-renewal ability) of epidermal stem cells or a regenerative medical product (processed cells, processed cell sheets, etc.).

[0070] Furthermore, one embodiment of the present invention relates to a pharmaceutical composition characterized by containing as an active ingredient a substance that resists molecular targeted drugs that impair the regenerative ability (self-renewal and maintenance) of epidermal stem cells, which substance induces or maintains the expression of COL17A1 in cells, suppresses the degradation of COL17A1 in cells, maintains the self-renewal ability of epidermal stem cells, suppresses genomic stress or oxidative stress in cells, and / or suppresses DNA damage in cells.

[0071] Furthermore, one embodiment of the present invention relates to a pharmaceutical composition for use in preventing or improving skin damage caused by anticancer agents, characterized in that it contains as active ingredients a substance that induces or maintains the expression of COL17A1 in cells, a substance that inhibits the degradation of COL17A1 in cells, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that inhibits genomic stress or oxidative stress in cells, and / or a substance that inhibits DNA damage in cells.

[0072] The pharmaceutical composition according to the present invention can be used for preventing or improving skin damage caused by anticancer drugs. In other words, one embodiment of the present invention relates to the use of a substance that induces or maintains the expression of COL17A1 in cells, a substance that suppresses the degradation of COL17A1 in cells, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that suppresses genomic stress or oxidative stress in cells, and / or a substance that suppresses DNA damage in cells in the manufacture of a medicine for use in preventing or improving skin damage caused by anticancer drugs.

[0073] Furthermore, one embodiment of the present invention relates to a pharmaceutical composition for use in anti-wrinkle treatment, characterized in that it contains as active ingredients a substance that induces or maintains the expression of COL17A1 in cells, a substance that inhibits the degradation of COL17A1 in cells, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that inhibits genomic stress or oxidative stress in cells, and / or a substance that inhibits DNA damage in cells.

[0074] The pharmaceutical composition of the present invention can be used for anti-wrinkle.In other words, one embodiment of the present invention relates to the use of a substance that induces or maintains the expression of COL17A1 in cells, a substance that inhibits the degradation of COL17A1 in cells, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that inhibits genomic stress or oxidative stress in cells, and / or a substance that inhibits DNA damage in cells in the manufacture of a medicament for anti-wrinkle.

[0075] Furthermore, one embodiment of the present invention relates to a pharmaceutical composition for use in improving rough skin, characterized in that it contains as active ingredients a substance that induces or maintains the expression of COL17A1 in cells, a substance that inhibits the degradation of COL17A1 in cells, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that inhibits genomic stress or oxidative stress in cells, and / or a substance that inhibits DNA damage in cells.

[0076] The pharmaceutical composition of the present invention can be used to improve rough skin. In other words, one embodiment of the present invention relates to the use of a substance that induces or maintains the expression of COL17A1 in cells, a substance that suppresses the degradation of COL17A1 in cells, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that suppresses genomic stress or oxidative stress in cells, and / or a substance that suppresses DNA damage in cells in the manufacture of a medicine for use in improving rough skin.

[0077] The pharmaceutical composition according to the present invention can be used for anti-age spots. In other words, one embodiment of the present invention relates to the use of a substance that induces or maintains the expression of COL17A1 in cells, a substance that inhibits the degradation of COL17A1 in cells, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that inhibits genomic stress or oxidative stress in cells, and / or a substance that inhibits DNA damage in cells in the manufacture of a medicine for use in anti-age spots.

[0078] In the pharmaceutical composition of the present invention, examples of substances that act as active ingredients include, but are not limited to, ROCK inhibitors, NADPH oxidase inhibitors, iNOS inhibitors, COX inhibitors, and ELANE inhibitors as described elsewhere in this specification, more specifically, Y27632, apocynin, and sivelestat.

[0079] The pharmaceutical composition according to the present invention can take any form such as a tablet, powder, liquid, or semisolid. The pharmaceutical composition according to the present invention may be an external medicine such as a liniment used for local treatment applied through the skin or directly to a skin lesion, and may be prepared by blending various main ingredients with a base. In addition to the above-mentioned active ingredient, the pharmaceutical composition according to the present invention may contain pharma- ceutically acceptable excipients, additives, buffers, salts for adjusting isotonicity, antioxidants, preservatives, drug stabilizers, and the like. Examples of excipients include, but are not limited to, water, purified water, alcohol, glycerin, lactose, starch, dextrin, sucrose, precipitated silica, honey, rice starch, and tragacanth. In addition, the pharmaceutical composition according to the present invention may contain other active ingredients. The blending amount of each ingredient can be appropriately determined within a medicament-acceptable range. In addition, the dosage of the composition can be appropriately determined depending on the type of drug used and the subject to be administered. For example, the active ingredient can be 0.01 to 15% by weight, for example, 0.1 to 5% by weight.

[0080] The route of administration can also be appropriately determined depending on the type of drug used and the subject to which it is administered. The pharmaceutical composition of the present invention may be contained in a wound covering material such as a dressing material. A dressing material is a medical material that can maintain a moist environment and provide an optimal environment for wound healing.

[0081] One embodiment of the present invention relates to a method for promoting skin wound healing in a mammal, a method for preventing or ameliorating skin ulcers or bedsores, a method for inhibiting skin aging, a method for enhancing the regenerative ability of epidermal stem cells, a method for preventing or ameliorating skin damage caused by anti-cancer drugs, an anti-wrinkle method, an anti-spot method, and / or a method for improving rough skin.

[0082] Such a method may include inducing or maintaining expression of COL17A1 in a cell, inhibiting degradation of COL17A1 in a cell, inhibiting genomic or oxidative stress in a cell, and / or inhibiting DNA damage in a cell. More specifically, one embodiment of the present invention may include administering, for example, a ROCK inhibitor, an NADPH oxidase inhibitor, an iNOS inhibitor, and / or a COX inhibitor to a mammal.

[0083] Examples of mammals include humans, monkeys, mice, rats, rabbits, dogs, cats, sheep, goats, alpacas, horses, cattle, pigs, minks, foxes, martens, raccoon dogs, chinchillas, sea otters, otters, beavers, and seals. The dosage can be appropriately determined depending on the type of drug used and the subject to which it is administered (as described above). The administration route can also be appropriately determined depending on the type of drug used and the subject to which it is administered. Preferred administration routes include, for example, application or spraying of a liquid, lotion, or cream to the skin, or subcutaneous injection of a liquid, or oral administration of a solid or liquid. A patch containing the composition according to the present disclosure may be prepared and applied to the skin.

[0084] Preventive and improving agent for pigmentation disorders One embodiment of the present invention relates to a method and composition for preventing or improving pigmentation abnormalities in aged skin. Such a method may include a step of inducing or maintaining the expression of COL17A1 in a cell, a step of suppressing the degradation of COL17A1 in a cell, a step of suppressing genomic or oxidative stress in a cell, and / or a step of suppressing DNA damage in a cell. Also, such a composition may include a substance that induces or maintains the expression of COL17A1 in a cell, a substance that suppresses the degradation of COL17A1 in a cell, a substance that suppresses genomic or oxidative stress in a cell, and / or a substance that suppresses DNA damage in a cell.

[0085] One embodiment of the present invention relates to a method and composition for preventing or improving wrinkles, fine lines, dryness, and sores due to skin aging. Such a method may include a step of inducing or maintaining COL17A1 expression in a cell, a step of suppressing degradation of COL17A1 in a cell, a step of suppressing genomic stress or oxidative stress in a cell, and / or a step of suppressing DNA damage in a cell. In addition, such a composition may include a substance that induces or maintains COL17A1 expression in a cell, a substance that suppresses degradation of COL17A1 in a cell, a substance that suppresses genomic stress or oxidative stress in a cell, and / or a substance that suppresses DNA damage in a cell.

[0086] Stem cell competition regulator One embodiment of the present invention relates to a method and composition for use in controlling cell competition in epidermal stem cells. The present inventors have found that COL17A1 is an important factor involved in cell competition. Thus, the method according to the present invention may include a step of inducing or maintaining the expression of COL17A1 in a cell, a step of suppressing the degradation of COL17A1 in a cell, a step of suppressing genomic stress or oxidative stress in a cell, and / or a step of suppressing DNA damage in a cell. In addition, the composition according to the present invention may include a substance that induces or maintains the expression of COL17A1 in a cell, a substance that suppresses the degradation of COL17A1 in a cell, a substance that suppresses genomic stress or oxidative stress in a cell, and / or a substance that suppresses DNA damage in a cell. It will also be understood by those skilled in the art that cell competition can be negatively controlled by inhibiting COL17A1.

[0087] Beauty supplements or functional foods One embodiment of the present invention relates to a beauty supplement or functional food containing a substance capable of maintaining the expression of COL17A1 in cells and the competitive self-renewal ability based thereon, a substance that suppresses the degradation of COL17A1 in cells, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that suppresses genomic stress or oxidative stress in cells, and / or a substance that suppresses DNA damage in cells. In one embodiment, the beauty supplement or functional food according to the present invention may be characterized by containing a substance that induces or maintains the expression of COL17A1 in cells as an active ingredient. More specifically, the beauty supplement or functional food according to the present invention may contain a ROCK inhibitor, an NADPH oxidase inhibitor, an iNOS inhibitor, and / or a COX inhibitor as a substance that induces or maintains the expression of COL17A1 in cells. More specifically, the beauty supplement or functional food of the present invention may contain, as substances that maintain the expression of COL17A1 in cells and maintain their self-replication ability, apocynin, ebselen, celecoxib, apigenin, Y-27632, ripasudil, 1400W, ethinyl estradiol, biochanin A, necrostatin 1, rafuma extract, mulberry extract, gymnema extract, tea extract, loquat leaf extract, milk thistle extract, black turmeric extract, psyllium oleracea seed extract, coffee seed extract, Somei-Yodoshino leaf extract, Melia azadirachta leaf extract, mandarin orange peel extract, lavender flower extract, Sophora angustifolia root extract, rice bran extract, Rosa canina fruit extract, Hamemaris leaf extract, eucalyptus leaf extract, hawthorn extract, and carrot extract.

[0088] The beauty supplement or functional food of the present invention may be in any form, such as a tablet, a powder, a semi-solid, a jelly or a liquid. The beauty supplement of the present invention may further include at least one of a skin anti-aging agent, a vitamin, collagen and a mineral. The beauty supplement or functional food of the present invention may be prepared, for example, to be taken orally 1 to 3 times a day before, during or after a meal.

[0089] Cosmetic Composition One embodiment of the present invention relates to a cosmetic composition, characterized in that it contains as active ingredients a substance capable of maintaining COL17A1 expression in cells and the competitive self-renewal ability based thereon, a substance that suppresses the degradation of COL17A1 in cells, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that suppresses genomic stress or oxidative stress in cells, and / or a substance that suppresses DNA damage in cells. As described elsewhere herein, examples of substances that induce or maintain COL17A1 expression in cells include, but are not limited to, ROCK inhibitors, NADPH oxidase inhibitors, iNOS inhibitors, and COX inhibitors. Examples of substances that induce or maintain the expression of COL17A1 in cells include apocynin, ebselen, celecoxib, apigenin, Y-27632, ripasudil, 1400W, astaxanthin, ethinyl estradiol, biochanin A, necrostatin 1, rafuma extract, mulberry extract, gymnema extract, tea extract, loquat leaf extract, milk thistle extract, black turmeric extract, psyllium oleracea seed extract, coffee seed extract, Somei-Yodoshino leaf extract, Melia azadirachta leaf extract, mandarin orange peel extract, lavender flower extract, Sophora angustifolia root extract, rice bran extract, Rosa canina fruit extract, Hamemaris leaf extract, eucalyptus leaf extract, and Crataegus crenata extract can be used.

[0090] In one embodiment, the cosmetic composition of the present disclosure is a composition for skin care.In one embodiment, the cosmetic composition of the present disclosure is a composition for application to the skin surface.The composition can be in various product forms, including but not limited to solutions, suspensions, lotions, creams, gels, toners, sticks, pencils, sprays, aerosols, ointments, cleansing liquids and bars, shampoos and hair conditioners, pastes, foams, powders, mousses, shaving creams, wipes, strips, patches, power patches, wound dressings and adhesive bandages, hydrogels, film-forming products, face and skin masks (with and without insoluble sheets), makeup products such as foundations, eyeliners and eyeshadows.

[0091] The cosmetic composition according to the present disclosure may contain 0.01 to 15% by weight, for example, 0.1 to 5% by weight, of a substance that induces or maintains the expression of COL17A1 in cells (Y27632, apocynin, etc.).

[0092] The cosmetic composition according to the present disclosure may further comprise at least one of a skin anti-aging agent, a skin tone agent, an anti-inflammatory agent, and a sunscreen agent.Skin anti-aging agents include, but are not limited to, peptides having anti-aging activity (WO2014157485A1).Suitable skin tone agents include, but are not limited to, sugar amines, arbutin, deoxyarbutin, hexylresorcinol, kojic acid, hexamidine compounds, salicylic acid, and retinoids including retinol propionate and retinyl propionate.

[0093] Suitable anti-inflammatory agents include, but are not limited to, nonsteroidal anti-inflammatory agents (NSAIDS such as ibuprofen, naproxen, etc.), glycyrrhizic acid (also known as glycyrrhetinic acid glycoside) and salts such as dipotassium glycyrrhizinate, glycyrrhetenic acid, licorice extract, bisabolol (e.g., alpha bisabolol), manjistha (extracted from plants of the genus Rubia, particularly Rubia cordifolia), and guggal (extracted from plants of the genus Commiphora, particularly Commiphora mukul), kola extract, chamomile, red clover extract, and whip coral extract (extract from plants of the order Gorgonian), derivatives of any of the above, and mixtures thereof.

[0094] Suitable sunscreens include 2-ethylhexyl-p-methoxycinnamate, 4,4'-t-butylmethoxydibenzoylmethane, 2-hydroxy-4-methoxybenzophenone, octyldimethyl-p-aminobenzoic acid, digalloyl trioleate, 2,2-dihydroxy-4-methoxybenzophenone, ethyl-4-(bis(hydroxypropyl)aminobenzoate, 2-ethylhexyl-2-cyano-3,3-diphenylacrylate, 2-ethylhexyl salicylate, glyceryl-p-amino Benzoate, 3,3,5-tri-methylcyclohexyl salicylate, methyl anthranilate, p-dimethyl-aminobenzoic acid or aminobenzoate, 2-ethylhexyl-p-dimethylaminobenzoate, 2-phenylbenzimidazole-5-sulfonic acid, 2-(p-dimethylaminophenyl)-5-sulfonebenzoxazoic acid, octocrylene, zinc oxide, benzylidene camphor and its derivatives, titanium dioxide, and mixtures thereof.

[0095] The cosmetic composition according to the present disclosure may further comprise at least one additive, such as a thickener, a fragrance, a pearlescent agent, a preservative, a sunscreen, an anionic or nonionic or cationic or amphoteric polymer, a protein, a protein hydrolysate, such as 18-methyl eicosanoic acid, a vitamin, panthenol, a silicone, a fatty acid such as a vegetable oil, an animal oil, a mineral oil or a synthetic oil, a gelling agent, an antioxidant, a solvent, a filter, a scrub ... Cosmetic ingredients such as cleaning agents, odor absorbers, colorants, abrasives, absorbents, fragrances, dyes, pigments / colorants, essential oils, anti-caking agents, defoamers, antimicrobial agents, binders, biological additives, buffers, fillers, chelating agents, chemical additives, cosmetic astringents, cosmetic biocides, denaturants, drug astringents, emollients, topical analgesics, film formers or materials, opacifiers, pH adjusters, preservatives, propellants, reducing agents, scavengers, skin cooling agents, skin protective agents, thickeners, viscosity regulators, vitamins, and combinations thereof may also be included. These additives may be present in the composition according to the present disclosure in a proportion that is not limited, but is preferably or advantageously in the range of 0 to 50% by weight, 5 to 40% by weight, or 30 to 50% by weight, based on the total weight of the composition.

[0096] The cosmetic compositions according to the present disclosure, for topical application to the skin, may in particular have the form of aqueous or oily solutions, dispersions of lotion or serum type, emulsions with a liquid or semi-liquid consistency of milk type obtained by dispersion of a fatty phase in an aqueous phase (O / W) or vice versa (W / O), suspensions or emulsions with a soft consistency of aqueous or anhydrous gel or cream type, or else microcapsules or microparticles, vesicular dispersions of ionic and / or non-ionic type, or foams. These compositions are prepared according to the usual methods. The compositions preferably have an aqueous phase between 5 and 99.5%.

[0097] The pH of the cosmetic composition according to the present disclosure is not limited, but is generally between 2 and 12, preferably between 3 and 9. The pH can be adjusted to the target value by adding a base (organic or inorganic) to the composition, for example, ammonia, sodium hydroxide, potassium hydroxide, or a primary, secondary or tertiary (poly)amine, such as monoethanolamine, diethanolamine, triethanolamine, isopropanolamine or 1,3-propanediamine, or with a basic amino acid or polyamino acid, such as lysine or arginine, or by adding an inorganic or organic acid, preferably a carboxylic acid, such as, for example, citric acid.

[0098] The cosmetic compositions according to the present disclosure in particular constitute creams (e.g. day creams, night creams, anti-aging creams, moisturizing creams, make-up removal creams, foundation creams or sun creams) for washing, protecting, treating or caring for the face, hands, feet, major anatomical folds or the body, liquid foundations, make-up removal milks, protective or caring body milks, sun milks, lotions, gels or foams for caring for the skin, such as cleansing lotions, sun lotions, artificial tanning lotions, bath compositions, deodorant compositions containing a disinfectant, aftershave gels or lotions, depilatory creams, compositions for combating insect bites.

[0099] The cosmetic composition according to the present disclosure may be applied at least once a day, twice a day, or more frequently during the treatment period. If applied twice a day, the first and second applications are separated, for example, by at least 1-12 hours. Typically, the composition may be applied in the morning and / or at night before going to bed.

[0100] Screening Methods The present invention relates to a method for screening for substances effective in promoting skin wound healing or preventing or improving skin ulcers, substances effective in inhibiting skin aging, substances effective in controlling cell competition, improving (enhancing, enhancing) the regenerative ability of epidermal stem cells, preventing or improving skin disorders caused by anticancer drugs, anti-wrinkles, anti-blemishes, and / or improving rough skin. Here, the inhibition of skin aging may be the inhibition of aging of hair follicles, which are skin appendages. Such a screening method may include the following steps i to iii: i) contacting a cell with a test substance in vitro; ii) measuring the expression of COL17A1 in the cells; and iii) determining whether the test substance increases the expression of COL17A1. Here, the measurement of COL17A1 expression can be performed using any method known to those skilled in the art. The substance identified by the above screening method is useful as a component for use in promoting skin wound healing, a component for use in suppressing skin aging, a component for use in controlling cell competition, a component for enhancing the regenerative ability of epidermal stem cells, a component for preventing or improving skin damage caused by anticancer drugs, an anti-wrinkle component, an anti-spot component, and / or a component for improving rough skin according to the present invention. It is also useful as a substance administered to a mammal in the method for promoting skin wound healing in a mammal, the method for suppressing skin aging, the method for enhancing the regenerative ability of epidermal stem cells, the method for preventing or improving skin damage caused by anticancer drugs, the anti-wrinkle method, the anti-spot method, and / or the method for improving rough skin according to the present invention.

[0101] The above screening method may further include a step of determining whether or not the test substance promotes colony formation. By combining the evaluation of COL17A1 expression with a colony formation assay, an agent that promotes "stem cell expansion via stem cell competition" can be screened by evaluating an agent that maintains self-renewal ability by maintaining continuous expression of COL17A1. That is, in one embodiment, the screening method may include the following steps i to iv: i) contacting a cell with a test substance in vitro; ii) measuring the expression of COL17A1 in the cells; iii) determining whether the test substance increases the expression of COL17A1; and iv) determining whether the test substance promotes colony formation.

[0102] Method and kit for evaluating skin aging One embodiment of the present invention relates to a method for evaluating skin aging using the expression of COL17A1 in cells as an index. The present inventors have found that the expression of COL17A1 is reduced in aged epidermal cells. Therefore, COL17A1 can be used as a marker for evaluating skin aging, and skin aging can be evaluated by measuring the expression of COL17A1 in epidermal cells. The expression of COL17A1 in epidermal cells can be measured by any method known to those skilled in the art, such as Western blotting or antibody staining using an antibody against COL17A1 (anti-COL17A1 antibody), or measuring mRNA by RT-PCR of the gene encoding COL17A1. The measurement results can be compared with a control or compared over time to evaluate skin aging. For example, if the expression level of COL17A1 is reduced as a result of the comparison, it is determined that skin aging is progressing.

[0103] Moreover, one embodiment of the present invention relates to a kit for use in a method for evaluating skin aging using the expression of COL17A1 in cells as an index. The kit includes, for example, an antibody that specifically recognizes COL17A1. Such an antibody may be directly labeled or may be recognized by a labeled secondary antibody. The label may be based on, for example, color development or chemiluminescence due to enzyme activity. The kit may also include, for example, an oligonucleotide (probe, primer) for detecting a transcription product of COL17A1. Such an oligonucleotide can be used in detection by hybridization with the transcription product or in an amplification reaction such as PCR. Thus, one embodiment of the present invention relates to a kit for use in a method for evaluating skin aging, including an antibody for detecting COL17A1 (anti-COL17A1 antibody) and a probe or primer for a gene encoding COL17A1. Furthermore, the kit may include components (antibodies, probes, primers, etc.) for measuring the expression levels of other marker proteins such as p16Ink4a.

[0104] Method and kit for evaluating epidermal stem cells One embodiment of the present invention relates to a method for evaluating epidermal stem cells using the expression of COL17A1 in cells as an index. The present inventors have found that there is a correlation between the proliferation ability of epidermal stem cells and the expression level of COL17A1. Therefore, COL17A1 can be used as a marker for evaluating epidermal stem cells, and epidermal stem cells can be evaluated by measuring the expression of COL17A1 in epidermal stem cells. Epidermal stem cells expressing COL17A1 at high levels have high proliferation ability, and can be evaluated as cells suitable for use in culture. The expression of COL17A1 in epidermal stem cells can be measured by any method known to those skilled in the art, such as Western blotting using an anti-COL17A1 antibody, antibody staining, ELISA, and FACS. The present inventors have also found that the expression of COL17A1 is reduced in aged epidermal stem cells. Therefore, the aging of epidermal stem cells can be evaluated by measuring the expression level of COL17A1 in epidermal stem cells. The aging of epidermal stem cells refers to the phenomenon in which the inherent self-renewal ability of epidermal stem cells is reduced due to aging, as well as various genomic and oxidative stresses, and they become more susceptible to being lost through terminal differentiation. For example, if the expression level of COL17A1 is deemed high as a result of comparison with the standard value, the cells are deemed suitable for use in culture or regenerative medicine products.

[0105] Moreover, one embodiment of the present invention relates to a kit for use in a method for evaluating epidermal stem cells using the expression of COL17A1 in cells as an index. The kit may contain, for example, an antibody that specifically recognizes COL17A1, a probe for a gene encoding COL17A1, or a primer for amplifying a gene encoding COL17A1. Thus, one embodiment of the present invention relates to a kit for use in a method for evaluating epidermal stem cells, comprising an antibody, a probe, or a primer for detecting COL17A1.

[0106] Method and kit for selecting epidermal stem cells One embodiment of the present invention relates to a method for selecting epidermal stem cells using the expression of COL17A1 in cells as an index. The present inventors have found that there is a correlation between the proliferation ability of epidermal stem cells and the expression level of COL17A1. Epidermal stem cells with high expression levels of COL17A1 have high proliferation ability and can be said to be suitable cells for use in culture. Therefore, COL17A1 can be used as a marker for evaluating the proliferation ability of epidermal stem cells, and measuring the expression of COL17A1 in epidermal stem cells and selecting epidermal stem cells with high expression levels of COL17A1 can be useful in subsequent culture or transplantation. Cells that highly express COL17A1 can be selected by any method known to those skilled in the art, for example, a method using FACS.

[0107] Furthermore, one embodiment of the present invention relates to a kit for use in a method for selecting epidermal stem cells using the expression of COL17A1 in cells as an index. The kit may contain, for example, an antibody, probe, or primer that specifically recognizes COL17A1, as described above. Thus, one embodiment of the present invention relates to a kit for use in a method for selecting epidermal stem cells, comprising an antibody, probe, or primer for detecting COL17A1.

[0108] Method for expanding epidermal stem cells and / or epidermal cells and evaluation kit One embodiment of the present invention also relates to a method for expanding epidermal stem cells and / or epidermal cells, comprising a step of selecting epidermal stem cells with high COL17A1 expression. Epidermal stem cells with high COL17A1 expression have high proliferation ability, so by selecting such cells, epidermal stem cells and / or epidermal cells can be expanded efficiently. Epidermal stem cells with high COL17A1 expression mean that the expression level is quantitatively higher than that in cells with advanced senescence. For example, they can be isolated by a method using FACS or a method using beads. Using such an expansion method, a cell sheet can be produced ex vivo.

[0109] Furthermore, one embodiment of the present invention relates to a kit for use in a method for expanding epidermal stem cells and / or epidermal cells, which includes a step of selecting epidermal stem cells that express high amounts of COL17A1. The kit may contain, for example, an antibody, probe, or primer that specifically recognizes COL17A1, as described above. Thus, one embodiment of the present invention relates to a kit for use in a method for expanding epidermal stem cells and / or epidermal cells, which contains an antibody, probe, or primer for detecting COL17A1.

[0110] Method and kit for evaluating cell competition One embodiment of the present invention relates to a method for evaluating molecules involved in cell competition, substances that promote or weaken the cell competition, and substances that change the efficiency or speed of the competition by changing the expression level of a molecule that modifies the expression of molecules involved in cell competition such as COL17A1 in some cells. The present inventors have found that the expression level of COL17A1 correlates with cell competition in epidermal stem cells. Epidermal stem cells with high expression levels of COL17A1 are advantageous in cell competition. In addition, cell competition can be evaluated using three-dimensional culture of epithelial cells that form stratified squamous epithelium, such as keratinocytes (including cultured keratinocytes, keratinocyte cell lines HaCat cells and Pam212 cells). Cells with high expression levels of COL17A1 can be evaluated as having high cell competitiveness because they grow more preferentially than cells with low expression levels of COL17A1 compared to the cells.

[0111] Furthermore, in the present invention, cell competition can be evaluated in a three-dimensional culture of keratinocytes, and compounds, extracts, nucleic acids, etc. that control this can be searched for. Thus, one embodiment of the present invention relates to a method for evaluating cell competition in keratinocytes, and in particular to a screening method for a substance that controls cell competition, using cell competition in a three-dimensional culture of keratinocytes (strain) as an index.

[0112] Moreover, one embodiment of the present invention relates to a kit for use in a method for evaluating cell competition in keratinocytes. The kit may include, for example, a nucleic acid that changes the expression of a cell competition-related molecule such as COL17A1 or a cancer-related gene, for example, a nucleic acid for use in RNA interference (such as siRNA or shRNA for a gene encoding COL17A1), a gene transfer reagent, a lentivirus or retrovirus, an antibody for immunostaining such as an antibody that specifically recognizes COL17A1, and a device for three-dimensional culture. Thus, one embodiment of the present invention relates to a kit for use in a method for evaluating cell competition in keratinocytes, including a cell line, a virus, an antibody, and a device involved in the evaluation of cell competition in keratinocytes. The kit may also include a keratinocyte cell line (for example, HaCaT cells) for performing three-dimensional culture.

[0113] cell composition One embodiment of the present invention relates to a cell composition (cell population) containing isolated cells expressing COL17A1. Cells expressing COL17A1 can be isolated by any method known to those skilled in the art, for example, FACS or a method using beads. The cell composition of the present invention may also contain cells having a three-dimensional structure, such as an epithelial sheet or an organoid.

[0114] The cells in the cell composition desirably express COL17A1 at a high level. Thus, the cell composition of the present invention includes cells that have been treated with a substance that induces or maintains the expression of COL17A1 in the cells. high The cells can be isolated, for example, by FACS. Here, the terms "high" and "hi" that indicate the expression level are well known in the art, and the term "COL17A1 high" indicates that the expression level of COL17A1 is at a high level in comparison with the cell population being analyzed. In the context of the present disclosure, "high" or "hi" may be considered to indicate that the expression level is within the top 50%, for example, within 3%, 5%, 10%, 15%, 20%, 30%, or 40%, of the cell population being analyzed. The cells in the cell composition may be, for example, epidermal keratinocytes or mucosal epithelial keratinocytes, but may also contain other cells. The cells in the cell composition may be, for example, cells in which at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the cells express COL17A1. The cell composition may contain at least 1×10 3 pieces, 1×10 4 pieces, 1×10 5 pieces, 1×10 6 pieces, 1×10 7 pcs or 1×10 8 The cell may include.

[0115] The cell composition may be a cell composition for use in transplantation. The cell composition may be a cell composition for use in treating skin disease or injury. The cells in the cell composition may be stem cell-derived cells. The cells in the cell composition may be cells in which COL17A1 expression has been induced or maintained using a substance that induces or maintains COL17A1 expression in cells. The substance that induces or maintains COL17A1 expression in cells may be at least one of a ROCK inhibitor, an NADPH oxidase inhibitor, an iNOS inhibitor, and a COX inhibitor. The substance that induces or maintains COL17A1 expression in cells may be Y27632 or apocynin.

[0116] The cells in the cell composition may be frozen. The cell composition may be contained in a single container.

[0117] Cell Sheet One embodiment of the present invention relates to a cell sheet comprising cells expressing COL17A1. Such a cell sheet can be produced by isolating and culturing cells expressing COL17A1. Such a cell sheet can be transplanted into a patient for use in treating a disease or injury in the patient. The cell sheet can be for autologous or allogeneic transplantation.

[0118] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these in any way. EXAMPLES

[0119] Example 1: Decrease in COL17A1 protein expression in aging skin Mouse tail skin, unlike dorsal skin, is characterized by stratified epidermal cell layers, as observed in histological analysis of human epidermis of epidermal melanocytes, and atrophy with a reduced number of spinous cell layers in the scaly epidermis. Basal layer cells in young mice have a rectangular and longitudinal shape relative to the basement membrane, whereas aged basal layer cells have a more rounded or flattened morphology. In aged skin, the total number of basal layer cells as well as the number of MCM2 cells are reduced. + A significant decrease in non-quiescent basal cells was observed, indicating that stem cells and their cell division in the epidermis decrease with age. Ultrastructural analysis of epidermal basal layer cells and the epidermal-dermal junction using transmission electron microscopy (TEM) revealed that the number of electron-dense areas (hemidesmosomes; HDs) that anchor the basal cell layer to the basal lamina significantly decreases with age. In addition, cell detachment from basal layer cells is frequently observed in the lamina lucida (LL) of aged tail skin, which is associated with a decrease in HDs from the basal cell layer with age.

[0120] Therefore, the inventor hypothesized that HD components are directly involved in the skin aging process. To test this, we analyzed the expression of various HD components and the major cell adhesion molecule, integrin β1 (ITGB1), in the tail epidermis of young and old (weeks) mice. As a result, it was revealed that the immunostaining level of COL17A1 was significantly decreased with aging (Figure 1). In agreement with this, immunoelectron microscopy analysis revealed that the COL17A1 signal in the LL of the dermal-epidermal junction in aged skin was significantly reduced. In addition, whole-body immunostaining revealed a heterogeneous distribution of COL17A1 and ITGA6 in aged skin. In particular, COL17A1-negative cell areas expressing ITGA6 were found in addition to double-negative cell areas in the aged tail epidermis. This indicates that the expression of COL17A1 protein is the most unstable, and as a result, insufficient COL17A1 ultimately destabilizes other HD components with aging.

[0121] To investigate the role of COL17A1 in the stability of HD components, we analyzed the number of HDs in young and aged mouse skin (Figure 2). Furthermore, COL17A1 proteolysis is also induced by persistent DNA damage responses, such as X-ray irradiation in mice and genomic instability in TTD progeria mice. Similarly, ultraviolet light (UV) irradiation also down-regulates COL17A1 expression in some basal layer keratinocytes in vitro and in vivo. As a result, UV irradiation of skin caused HD destabilization. These data together suggest that COL17A1 functions as a stress / fitness sensor that monitors the DNA damage response as a "stem cell checkpoint molecule" and determines whether stem cells self-renew or differentiate. This stress-sensing mechanism may cause differences in the self-renewal capacity of epidermal stem cell clones with different COL17A1 expression levels present in the aged basal layer of the epidermis.

[0122] Example 2: COL17A1 in the skin aging process + Clonal expansion of epidermal stem cells To visualize the dynamics of epidermal stem cell clones that emerge with aging and to track the fate of COL17A1-deficient clones, we generated a basal layer keratinocyte lineage tracing system by drug-inducible multicolor labeling in K14-CreERT2;R26R Brainbow 2.1 mice. This system allows stochastic multicolor labeling of basal stem / progenitor cells by Cre-LoxP-mediated recombination. After tamoxifen (TAM) administration, GFP-, RFP-, YFP- or CFP-expressing cells were found stochastically in the tail skin of young (4.5 months old) mice. In contrast, the area of ​​single-colored keratinocyte clones significantly increased with aging, accompanied by a decrease in the total number of clones (Figure 3), indicating that some stem cells undergo clonally expanding at the expense of many other cells during physiological aging.

[0123] Next, to test the possibility that heterogeneity of basal cells with different expression levels of COL17A1 induces stem cell competition in the basal layer during aging, we observed the expression levels of COL17A1 by multicolor-labeled stem cell clones in the tail interfollicular epidermis (IFE) of young (11-month-old) and old (28-month-old) mice. As shown in Figure 4, COL17A1 expression in monochromatic basal layer cells representing stem cell clones was generally decreased in aged skin, whereas the expression of COL17A1 in basal layer cells of young mice was generally decreased with aging, with each clone having a different COL17A1 expression level. A detailed comparison of the clone size and COL17A1 expression level of basal layer keratinocytes revealed that the clone size of basal layer cells was positively correlated with the expression level of COL17A1 in aged mouse skin (Figure 5). This indicates that stem cell clones that maintain higher levels of COL17A1 expression exhibit higher clonogenicity and become predominant in the basal layer of the epidermis during the physiological aging process. Furthermore, there was no significant induction of apoptotic cell death or so-called "senescent" cells in basal layer cells of aged skin. These data support the conclusion that COL17A1 expression is a potent marker of aging. high Epidermal stem cells are COL17A1 low This suggests that they compete with cells to resist epidermal aging.

[0124] Example 3: Differential expression of COL17A1 leads to competition for epidermal stem cells COL17A1 low / - The emergence of clones was confirmed by the in vivo analysis of the adjacent COL17A1 high To investigate whether Col17a1 expression directly induces cell competition with clones, we generated drug-inducible Col17a1 gene knockout (cKO) mice combined with a multicolor labeling system specific for basal layer keratinocytes. Rare induction of COL17A1 deficiency using low-dose TAM administration resulted in monochromatic labeled Col17a1-deficient keratinocyte clones in the epidermis of the tail 2 days after knockout induction. In control mice, these clones increased in size at day 14 and then formed monochromatic columns (basal clones) at day 28. In contrast, many Col17a1-deficient basal layer keratinocyte clones were surrounded by unlabeled basal layer keratinocyte clones with high levels of COL17A1 expression, which detached around day 14 and became loser cell clones (floating clones) by day 28 (Figure 6). Consistent with this, whole-mount analysis of mouse tails revealed a marked elimination of Col17a1-deficient cell clones from the skin. These data indicate that COL17A1-expressing epidermal stem cells compete with COL17A1-deficient epidermal stem cells in the niche and that COL17A1 mediates skin homeostasis and the ageing process through a cell competition mechanism in the epidermis.

[0125] To further investigate whether differential expression of COL17A1 directly mediates cell competition in the epidermis, we established a novel in vitro cell competition assay system by expressing short hairpin (sh)RNA in 3D-cultured HaCaT human keratinocytes. + COL17A1 - Stable HaCaT cells expressing shScramble as well as control EmGFP + COL17A1 +HaCaT cells were generated and cultured in a 3D culture system. Both shScramble and shCOL17A1-expressing HaCaT cells showed no obvious differences in the structure, stratification, proliferation number, or apoptotic cells in the 3D cultured epidermis. - EmGFP + Cells and wild-type (COL17A1 + ) cells at a ratio of 1:10 significantly eliminated shCOL17A1-expressing cells. However, no significant elimination of COL17A1-expressing cells was detected at a ratio of 1:3. These data indicate that Col17a1-deficient basal layer cells are eliminated from the basal layer as loser cells by a sufficient number of surrounding wild-type cells. Collectively, these data suggest that COL17A1-mediated cell competition occurs during the physiological aging process of human epidermis and that COL17A1 expressed by epidermal stem cells confers competitive self-renewal potential between neighboring epidermal stem cell populations.

[0126] Example 4: Maintenance of epidermal homeostasis by COL17A1-mediated SCD To understand the mechanism of COL17A1-mediated cell competition, the present inventors investigated whether COL17A1 regulates cell competition by inhibiting COL17A1 + We hypothesized that Col17a1 promotes SCD, which theoretically maintains the clonal expansion of epidermal stem cells. To test this hypothesis, we analyzed the division axis of basal layer cells in vivo. Whereas normal basal layer cells showed cell divisions mostly parallel to the basement membrane, Col17a1-deficient basal layer keratinocytes abnormally induced perpendicular cell divisions to generate basal layer cells and progeny layers located above the basal layer. This indicates that COL17A1 mediates SCD and suggests that COL17A1-mediated SCD pushes Col17a1-deficient basal layer cells, which are lost or reduced in HD, away from the basement membrane. Indeed, Col17a1-deficient keratinocytes eventually detach from the basal layer IFE.

[0127] Thus, these data support the conclusion that COL17A1 +We show that COL17A1-dependent SCD, which promotes clonal expansion of winner cells, drives cell competition during the aging process. Importantly, basal layer cells in aged epidermis show an increased proportion of vertical cell divisions and often show reduced levels of COL17A1 expression. This ultimately leads to epidermal atrophy (thinning) due to impaired cell division with loss of COL17A1 expression in aged skin regions. In sharp contrast, epidermal basal layer cells in COL17A1 transgenic (tg) mice, whose basal layer cells maintain COL17A1 expression, maintained cell division horizontal to the basement membrane. These data indicate that COL17A1-mediated SCD generates a mechanical driving force for cell competition and endures epidermal aging through competitive self-renewal phenomena, resulting from horizontal cell spreading and basement membrane area occupation in adult epidermis.

[0128] It is known that in hematopoietic stem cells, the amplification of certain stem cell clones occurs predominantly, while other stem cell clones disappear, and that the self-renewal of stem cells occurs competitively (competitive self-renewal). The molecules involved in this phenomenon are also gradually being clarified. However, the actual state of "competitive self-renewal" in the epidermis under physiological conditions was not known, and the molecules responsible for this had not been clarified.

[0129] To test whether COL17A1-mediated SCD sustains clonal expansion of epidermal stem cell clones, we used a colony formation assay, the gold standard in vitro assay for assessing the self-renewal potential of epidermal stem cells, including cells in tail epidermis. Senescent epidermal keratinocytes expressing low levels of COL17A1 exhibited reduced colony-forming potential and generated smaller colonies compared to younger tail keratinocytes (Figure 7). In contrast, forced expression of hCOL17A1 in mouse basal layer keratinocytes promoted the clonogenic potential of epidermal stem cells, with colony size and number significantly greater than in control mice (Figure 8).

[0130] These data indicate that COL17A1 mediates clonal expansion through the proliferative capacity of epidermal stem cells. Consistent with this, expression of COL17A1 in tg mice significantly rescued age-associated epidermal thinning and age-associated desquamation. Taken together, the COL17A1-dependent proliferative capacity of epidermal stem cells undergoing continuous symmetric cell division (SCD) in colony assays supports the role of COL17A1 in epidermal homeostasis and cell competition during aging. high This explains the higher fitness of stem cell clones and they are said to have competitive self-renewal capacity.

[0131] Example 5: Maintenance of heterotypic cell lineages by COL17A1-expressing epidermal stem cells Skin aging is characterized not only by epidermal aging but also by melanocyte-associated pigmentation abnormalities and dermal changes. To study the functional significance of epidermal stem cell aging in skin aging, we assessed whether physiological aging induces epidermal pigmentation abnormalities in the tail epidermis. We observed that physiological aging gradually induces skin pigmentation abnormalities (uneven skin pigmentation with areas of low and high pigmentation) and eventually induces hypopigmentation in the tail skin. Detailed microscopic observation of tail whole mounts revealed that the pigmentation pattern becomes uneven with age. To visualize the distribution of melanocyte lineage cells in the epidermis, we analyzed the tail skin of Dopachrome tautomerase (Dct) promoter-controlled histone-H2B GFP (Dct-H2B GFP) tg mice. Section and whole mount analysis showed that H2B GFP was significantly increased in the tails of young mice. + Epidermal melanoblasts / melanocytes were observed, but these cells were shown to disappear in an age-dependent manner in the epidermis of aged tails. Consistent with this, microarray analysis of young and aged whole epidermal cells ranked melanocyte genes as the top major changes during aging. Importantly, highly pigmented melanocytes were observed in the basal region of 12-month-old tails, and these cells were occasionally present in the suprabasal region, suggesting that they were delaminating from the basal layer and being eliminated from the skin.

[0132] Next, to investigate whether stem cell senescence of epidermal keratinocytes induces changes in epidermal melanocytes, we examined whether loss of HD components by Col17a1 and / or Itga6 deficiency in epidermal basal cells induces early skin pigmentation abnormalities. Time-course analysis revealed that Col17a1 deficiency induces relatively mild pigmentation abnormalities by inducing HD instability, while Col17a1 and Itga6 genes synergistically induce significant pigmentation abnormalities. Indeed, epidermal melanocytes tagged with Dct-H2B GFP were depleted from skin upon Col17a1 or Itga6 deficiency.

[0133] Chronic UVB irradiation induces skin pigmentation abnormalities and HD damage in mouse and human epidermis. We next examined whether chronic UVB exposure induces skin pigmentation abnormalities and HD damage, as well as its associated phenotypes in the tail epidermis (Figure 9). Following skin hyperpigmentation induced by repeated UVB irradiation, epidermal pigmentation abnormalities were induced within 1 month after the final UVB irradiation, similar to Col17a1 or ITGA6 cKO mice. Ultrastructural analysis using TEM and immunoTEM revealed that UVB irradiation inhibited the formation of mature HD and reduced the signal of COL17A1, similar to Col17a1 cKO.

[0134] Together, these data indicate that chronic UVB-mediated HD damage induces epidermal dyspigmentation through the impairment of COL17A1-expressing epidermal keratinocytes. Moreover, overexpression of COL17A1 in basal layer keratinocytes in mice leads to macroscopic and microscopic dyspigmentation phenotypes, as well as KIT + COL17A1 significantly rescued the age-related decline in epidermal melanocytes (Figure 9). Collectively, these data indicate that melanocytes in the epidermis are maintained by neighboring COL17A1-expressing epidermal stem cells in the epidermis during chronological and photoaging.

[0135] The regenerative capacity of skin declines with age. We examined whether age-related changes in epidermal stem cells are involved in skin wound healing, which proceeds via epidermal stem cell cloning, and whether they affect dermal fibroblasts (Figure 10). Analysis of PDGFRα-positive mesenchymal cells showed that the population located just beneath the epidermis was significantly reduced with age, and forced expression of hCOL17A1 rescued the age-related loss of PDGFRα-positive cells. These data suggest that COL17A1-mediated maintenance of epidermal stem cells enhances the stability of the epidermal-dermal junction and resists the aging of skin organs, including skin atrophy, fine wrinkles, and skin aging caused by its breakdown.

[0136] Example 6: HD is the key to skin organ regeneration To test the above hypothesis, we performed full-thickness wound healing experiments using tail skin of aged wild-type mice. Measurement of wound area showed that physiological aging significantly delayed the wound healing process. We then analyzed Col17a1 or ITGA6-deficient skin and found that both showed significantly delayed wound repair (Figure 11), indicating that HD instability leads to poor wound healing in physiological aging. Forced expression of hCOL17A1 by mouse basal layer keratinocytes also promoted wound healing (Figure 12). These data indicate that COL17A1-mediated clonal expansion of epidermal stem cells is essential not only for stem cell competition in skin homeostasis but also for the regeneration of full-thickness skin wound healing.

[0137] Since overexpression of COL17A1 promotes skin wound healing, the present inventors searched for novel compounds that induce COL17A1 expression in keratinocytes in vitro. As shown in Table 1 and Figure 15, the present inventors identified two chemicals, Y27632 and Apocynin, that induce COL17A1 expression in cultured keratinocytes. To confirm in vitro that the effects of these compounds are mediated by increasing the competitive self-renewal ability of epidermal stem cells exhibiting SCD, the present inventors performed a colony formation assay using human epidermal keratinocytes. Consistent with this, the number of colonies was significantly increased by Y-27632, and the colony size was significantly increased by both agents (Table 1, Figures 13 and 14).

[0138] To confirm the beneficial effects in vivo, these drugs were administered to full-thickness wounds in the tail skin of mice. Both drugs significantly promoted the wound repair process, similar to that in tg mice overexpressing human COL17A1 (Figure 12). These results indicate that COL17A1-inducing drugs promote skin wound healing through promoting re-epithelialization of the wound edge by epidermal stem cell proliferation, and these findings open a new avenue for new drugs to promote skin regeneration.

[0139] Example 7: Search for substances that maintain and promote the competitive self-renewal ability of epidermal stem cells We further explored substances that induce or maintain COL17A1 expression. Human epidermal keratinocytes were seeded on a 384-well plate, and cultured for 48 hours with reagents. After performing fluorescent immunostaining with anti-COL17A1 antibody and fluorescently labeled secondary antibody, the fluorescence intensity per cell was detected using a high-content imaging system (HCS: high-content screening). In addition, some of the substances were analyzed by Western blotting (WB). In addition to counting the number of cells using DAPI, the expression level of COL17A1 was quantified.

[0140] As a result, we identified a group of substances that maintain the self-renewal ability of epidermal stem cells through the expression of COL17A1. However, it is difficult to distinguish between substances that transiently increase COL17A1 expression, such as steroids and other cytotoxic substances, but attenuate self-renewal ability in the long term, and these cannot be excluded by measuring COL17A1 expression. Therefore, by combining this with a colony formation assay, we succeeded in selecting substances that promote COL17A1-mediated competitive self-renewal ability in the long term. The results are shown in Figure 15 and Tables 1 and 2.

[0141] [Table 1]

[0142] In Table 1, for COL17A1 expression level (relative to control), two double circles (◎◎) indicate an increase of 2.0 times or more in expression level, one double circle (◎) indicates an increase of 1.2 times or more, and a circle (〇) indicates maintenance of 1.0-1.2 times. For colony number or colony area relative to control, two double circles (◎◎) indicate an increase of 1.2 times or more, one double circle (◎) indicates maintenance of 1.0-1.2 times, and X indicates 1.0 or less (same below). ND (Not done) indicates unanalyzed.

[0143] Among the substances that induce or maintain the expression of COL17A1 expressed by epidermal stem cells, many were found to enhance colony formation ability, i.e., the self-renewal ability of epidermal stem cells (Figure 16, Tables 1 and 2). On the other hand, there were also substances that were difficult to distinguish from stress-responsive COL17A1 expression fluctuations, and substances that stably reduced colony formation ability. Therefore, by combining both COL17A1 expression analysis and colony formation ability, it is possible to select substances that have "competitive self-renewal ability" in vivo.

[0144] [Table 2]

[0145] [Table 3]

[0146] Anticancer drugs and molecular targeted drugs that reduce the expression level of COL17A1 are summarized in Table 3. COL17A1 expression fluctuates with conventional chemotherapy drugs such as metabolic antagonists and alkylating agents. In particular, COL17A1 expression is significantly reduced by molecular targeted drugs that target cancer cells (Table 3).

[0147] Example 8: Decreased COL17A1 levels due to anticancer drugs and various stresses Seven-week-old C57BL6 mice were administered the anticancer drug hydroxyurea three times a month, and tissues were collected three months later and immunostained using COL17A1 antibodies. As a result, the expression of COL17A1 in the basal layer was significantly reduced in mice administered hydroxyurea (Figure 17). In addition, human epidermal keratinocytes, HaCaT cells, were irradiated with radiation (20Gy, 30Gy) and ultraviolet light (20mJ, 30mJ), and the cells were collected 24 hours and 72 hours later, respectively, and Western blotting was performed using antibodies against COL17A1. As a result, a significant decrease in the amount of COL17A1 protein was observed in both radiation and ultraviolet light irradiation. Furthermore, when hydrogen peroxide, which induces oxidative stress, was administered to HaCaT cells and the expression of COL17A1 was analyzed in the same manner, a significant decrease was observed (Figure 17).

[0148] From the above, it became clear that the expression of COL17A1 is decreased by various types of stress, including anticancer drugs, ultraviolet light, radiation, and oxidative stress.

[0149] Example 9: Test to improve skin damage caused by anticancer drugs by overexpressing COL17A1 Transgenic mice overexpressing human COL17A1 (COL17A1-Tg) and control mice (approximately 18 months old) were acclimated to the experimental environment, and then the hair on their backs was removed under anesthesia and they were divided into the following six groups. Control mice not administered EGF receptor inhibitors (n=3) COL17A1-Tg mice not treated with EGF receptor inhibitors (n=3) Control mice treated with EGF receptor inhibitor (erlotinib) (n=4) COL17A1-Tg mice treated with EGF receptor inhibitor (erlotinib) (n=4) Control mice treated with EGF receptor inhibitor (gefitinib) (n=3) COL17A1-Tg mice treated with EGF receptor inhibitor (gefitinib) (n=3)

[0150] Erlotinib or gefitinib, a type of anticancer drug and an EGF receptor molecular targeting drug, was dissolved in 0.5% methylcellulose solution at a concentration of 5mg / ml and administered intraperitoneally at 50mg / kg / day every day from the start of the study. A 0.5% methylcellulose solution of the solvent was administered to the control group in the same way. Transepidermal water loss (TEWL) was measured for mice in each group on the 7th day after the start of erlotinib or gefitinib administration. Measurements were performed at specific sites on the back of the mice (two sites symmetrically on the left and right from the center of the spine) using a Tewameter TN300 (manufactured by Courage+Khazaka, Germany, Integral Co., Ltd.).

[0151] The test results are shown in Figure 18. The results in Figure 18 show that TEWL was significantly increased in control mice administered erlotinib or gefitinib, but in comparison, Tg mice overexpressing COL17A1 showed significantly lower TEWL values ​​even when administered erlotinib or gefitinib, suggesting that skin damage was alleviated. This shows that skin damage caused by the administration of anticancer drugs is significantly improved by promoting the self-renewal ability of epidermal stem cells through the overexpression of COL17A1.

[0152] Example 10: Test to improve skin damage caused by anticancer drug administration using a substance that promotes the self-renewal ability of epidermal stem cells Female C57BL6N mice (7 weeks or 6 months old) were acclimated to the experimental environment, and then the hair on their backs was removed under anesthesia. They were then divided into the following 6 groups (n=4) so ​​that their body weights were approximately uniform. 7 weeks old / non-EGF receptor inhibitor (erlotinib) group 7 weeks of age / EGF receptor inhibitor (erlotinib) administered + control vehicle applied group 7 weeks old / EGF receptor inhibitor (erlotinib) administration + apocynin 200 μM application group 6 months old / non-EGF receptor inhibitor (erlotinib) group 6 months old / EGF receptor inhibitor (erlotinib) administered + control vehicle applied group 6 months old / EGF receptor inhibitor (erlotinib) administered + apocynin 200 μM applied group

[0153] Erlotinib was dissolved in 0.5% methylcellulose solution at a concentration of 5mg / ml and administered intraperitoneally at 50mg / kg / day every day from the start of the study. A 0.5% methylcellulose solution of the solvent was administered in the same way to the control non-administration group. An aqueous solution of each preparation was applied to the back of the mice once a day, five times a week, to the control solvent application group and the apocynin 200 μM application group. The apocynin solvent (50% ethanol / PBS) was applied to the control solvent application group in the same way. TEWL was measured for mice in each group on the 7th day after the start of erlotinib administration for 7-week-old mice and on the 12th day for 6-month-old mice. Measurements were performed using a Tewameter TN300 (manufactured by Courage+Khazaka, Germany, Integral Co., Ltd.) at specific sites on the back of the mice (two sites symmetrically on the left and right from the center of the spine).

[0154] The test results are shown in Figure 19. As shown in Figure 19, TEWL was significantly increased in mice administered erlotinib, but in the group in which apocynin was applied, TEWL was significantly decreased and skin damage was alleviated, even under erlotinib administration. This shows that skin damage caused by the administration of molecular targeted drugs can be significantly improved by substances that promote the cell competitive ability of epidermal stem cells.

[0155] Example 11: Test to improve skin disorders in aged mice using a substance that promotes the self-renewal ability of epidermal stem cells A substance that promotes the cell competitive ability of epidermal stem cells (apocynin) was continuously applied to a model of irritant skin disorder (dry eczema and alopecia) induced by high-concentration ethanol (100%) in the neck-upper back skin of aged mice. As a result, as shown in Figure 20, the skin disorder was significantly alleviated, and a significant preventive and therapeutic effect was obtained.

[0156] Example 12: Barrier function improvement test The skin barrier function is the main function of the skin, and it prevents moisture from evaporating from the body and foreign substances from entering the body, but when the epidermis thickens reactively due to UV exposure, the barrier function is weakened. Generally, the stratum corneum moisture content and TEWL are used to evaluate the skin barrier function, and together with the reactive epidermal thickness, they are considered to be indicators that reflect the degree of skin damage caused by UV rays, anticancer drugs, etc.

[0157] Hairless mice HOS:HR-1 (female, 7 weeks old) were acclimatized to the experimental environment, and then divided into the following 6 groups so that the body weights were approximately uniform. · UV non-irradiation group (n=2) Ultraviolet (UVB) irradiation + solvent application group (n=4) UVB irradiation + apocynin 200 nM application group (n=4) · Ultraviolet (UVB) irradiation + apocynin 200 μM application group (n=4) · Ultraviolet (UVB) irradiation + Y-27632 2 μM application group (n=4) · Ultraviolet (UVB) irradiation + Y-27632 20 μM application group (n=4)

[0158] In the solution application group, the aqueous solution of each preparation was applied to the back of the hairless mice once a day, starting from the day before UV irradiation, for five days each day. In the "UV + solvent application group", the solvent (50% ethanol / PBS) of the preparation in Example 1 was applied in the same manner. UVB irradiation was performed using a Yayoi UV irradiation device (Y-UV-Lab-K-D 5-lamp type) at 150 mJ / cm once a day. 2The stratum corneum moisture content and TEWL of the irradiated area were measured for each group of mice on the fourth day after the start of UV irradiation. Measurements were performed at specific sites on the back of the mice (two sites symmetrically on the left and right sides of the spine) using a Corneometer CM825 and a Tewameter TN300 (manufactured by Courage+Khazaka, Germany, and Integral Corporation).

[0159] Figure 21 shows the effect of substances that promote the self-renewal ability of epidermal stem cells on maintaining stratum corneum moisture content in response to UV irradiation. The moisture content of the stratum corneum after UV irradiation was significantly lower than that of the control group without UV irradiation, but the groups administered 200 μM apocynin and 2 μM and 20 μM Y-27632 showed significantly higher moisture content. This demonstrates that substances that promote the cell competitive ability of epidermal stem cells have the effect of maintaining stratum corneum moisture content in response to UV irradiation. Furthermore, the TEWL suppression effect of substances that promote the self-renewal ability of epidermal stem cells against UV irradiation was analyzed. As shown in Figure 21, the TEWL after UV irradiation was significantly higher than that of the control group without UV irradiation, but the 200 μM apocynin administration group and the 20 μM Y-27632 administration group showed significantly lower TEWL. This demonstrated that substances that promote the cell competitive ability of epidermal stem cells have the effect of suppressing the increase in TEWL caused by UV irradiation.

[0160] Example 13: Evaluation of improvement in skin elasticity (skin firmness) When the condition of the dermis changes due to ultraviolet rays and aging, the skin loses elasticity (= firmness), causing wrinkles and sagging. Indicators of skin elasticity include the stretching of the skin when pulled and the return to its original state when released, and it is known that multiple elasticity indicators decrease with age. It is believed that an increase in skin elasticity leads to an improvement in wrinkles, sagging, and firmness of the skin.

[0161] Hairless mice HOS:HR-1 (female, 7 weeks old) were acclimatized to the experimental environment, and then divided into the following three groups (n=4) so ​​that the body weights were approximately uniform. · UV non-irradiation group UVA+UVB irradiation + solvent application group · Ultraviolet (UVA+UVB) irradiation + apocynin 200 μM application group

[0162] In the solution application group, an aqueous solution of each preparation was applied to the back once a day, three times a week, for five weeks, starting from the day before UV irradiation. In the "UV + solvent application group," the apocynin solvent (50% ethanol / PBS) was similarly applied. UVB irradiation was performed using a Yayoi UV irradiation device (Y-UV-Lab-K-D 5-lamp type), and UVA was applied three times a week at 2 J / cm. 2 , UVB 150mJ / cm 2 The irradiation was carried out for 5 weeks at an intensity of 100 nm.

[0163] Skin elasticity was measured on the back (two sites symmetrically on the left and right sides of the spine) of each group 5 weeks after the start of UV irradiation. Skin elasticity was measured using Cutometer MPA580 (Courage+Khazaka, Germany, Integral Co., Ltd.) (parameter settings: 300mba, on time 2 seconds, off time 2 seconds), and the average of three measurements was used for analysis. The skin on the back of the mouse was suctioned for 2 seconds, and then released after 2 seconds. The length of the skin stretched immediately after suction, immediately before suction release, and immediately after suction release were measured and used as the rapid extensibility at the beginning of suction (Ue: unit mm), the maximum skin extensibility immediately before suction release (Uf: unit mm), and the rapid recovery immediately after suction release (Ur: unit mm). R5=Ur / Ue was used as an index of net elasticity, and R7=Ur / Uf was used as an index of elasticity.

[0164] As shown in Figure 22, the skin elasticity value is significantly decreased by UV irradiation, but the test group to which the substance promoting the self-renewal ability of epidermal stem cells was applied showed a significant improvement in skin elasticity compared to the comparison group. It is believed that an increase in skin elasticity leads to an improvement effect on wrinkles, sagging, or firmness of the skin. This suggests that the application of a substance promoting the cell competitive ability of epidermal stem cells can have an effect of improving wrinkles, sagging, or firmness of the skin.

[0165] Example 14: Evaluation of skin elasticity (skin firmness) improvement by long-term application To investigate the effect of substances that promote the competitive ability of epidermal stem cells on improving skin firmness, female hairless mice HOS:HR-1 (7 weeks old) were acclimated to the experimental environment and then divided into the following three groups (n=2) so that their body weights were roughly uniform. Control (solvent) treatment group Apocynin 200μM application group Retinol 0.1% application group

[0166] In this case, the solution application group was applied with the aqueous solution of each preparation once a day, three times a week for seven weeks. Seven weeks after the start of application (day 53), TEWL and skin elasticity measurements were performed on the back (two symmetrical locations on the left and right sides of the spine center) of each group. TEWL measurements were performed on specific sites on the back of the mouse (two symmetrical locations on the left and right sides of the spine center) using a Tewameter TN300 (manufactured by Courage+Khazaka, Germany, Integral Co., Ltd.). Skin elasticity was measured using a Cutometer MPA580 (manufactured by Courage+Khazaka, Germany, Integral Co., Ltd.) (parameter settings: 300mba, on time 2 seconds, off time 2 seconds), and the average value of three measurements was used for analysis. The skin on the back of the mouse was suctioned for 2 seconds, and then released after 2 seconds. The stretched length of the skin was measured immediately after suction, immediately before suction was released, and immediately after suction was released. These were the skin's return when suction was released (Ua: unit mm), the rapid extensibility at the beginning of suction (Ue: unit mm), the maximum skin extensibility immediately before suction was released (Uf: unit mm), and the rapid recovery immediately after suction was released (Ur: unit mm). R5 = Ur / Ue was set as an index of net elasticity, and R7 = Ur / Uf was set as an index of elasticity. R2 = Ua / Uf was set as an index of total elasticity.

[0167] As shown in Figure 23, in the test group in which the substance that promotes the self-renewal ability of epidermal stem cells was applied continuously for a long period of time, skin elasticity was significantly improved in all three indices compared to the comparative retinol. Furthermore, while continuous application of retinol significantly increases the TEWL of the skin and is thought to have the effect of suppressing the barrier function, the substance that promotes the self-renewal ability of epidermal stem cells maintains normal barrier function, and it is thought that continuous application of the substance that promotes the self-renewal ability of epidermal stem cells increases elasticity while maintaining normal skin barrier function, and brings about the effect of preventing and improving wrinkles, sagging, and firmness of the skin.

[0168] Example 15: Wrinkle improvement test In a mouse model in which wrinkles were induced by long-term exposure to ultraviolet A radiation, a substance that promotes the self-renewal ability of epidermal stem cells was shown to improve wrinkles.

[0169] Hairless mice HOS:HR-1 (female, 7 weeks old) were acclimatized to the experimental environment, and then divided into the following three groups so that the body weights were approximately uniform. · UV non-irradiation group (n=2) UVA+UVB irradiation + solvent application group (n=4) UVA+UVB irradiation + apocynin 200μM application group (n=4) - UVA+UVB irradiation + 0.1% retinol application group (n=2)

[0170] In the solution application group, an aqueous solution of each preparation was applied to the back of hairless mice once a day, three times a week, for five weeks, starting from the day before UV irradiation. In the "UV + solvent application group," the apocynin solvent (50% ethanol / PBS) was applied in the same way. Retinol, which is said to be effective in improving wrinkles, was also applied in the same way as a control group at a concentration of 0.1%. UVB irradiation was performed using a Yayoi ultraviolet irradiation device (Y-UV-Lab-K-D 5-lamp type), with UVA at 20 J / cm three times a week. 2 , UVB 100mJ / cm 2 The irradiation was carried out for 4 weeks at an intensity of 100 nm. The backs of the mice in each group were photographed from 1 to 4 weeks after the start of UV irradiation using a high-resolution three-dimensional image analyzer PRIMOS-CR (Canfield Scientific, USA, Integral Corporation). Based on the images obtained, the total wrinkle volume (mm 3 ) and total average wrinkle depth (μm). Skin flexibility was measured using a Cutometer MPA580 (Courage+Khazaka, Germany; Integral Co., Ltd.), and the R0 value was taken as skin flexibility. The R0 value reflects the state of the keratin and is an index of skin flexibility. TEWL was measured on the back of each group (two locations symmetrically on the left and right sides of the spine) using a Tewameter TN300 (Courage+Khazaka, Germany; Integral Co., Ltd.).

[0171] As shown in Figure 24, UV exposure significantly increases skin wrinkles, but in the test group to which a substance that promotes the competitive ability of epidermal stem cells was applied, wrinkle formation was suppressed compared to the control group, and the degree of suppression was more pronounced than in the case of retinol. 3 ) and the total average wrinkle depth (μm) were measured, and as shown in Figure 24, skin wrinkles increased significantly due to UV exposure, but were significantly improved in the test group to which the substance that promotes the cell competitive ability of epidermal stem cells was applied. This suggests that the application of a substance that promotes the self-renewal ability of epidermal stem cells can have an effect on improving skin wrinkles.

[0172] In addition, when the transepidermal water loss (TEWL) was measured for each group of mice, the group to which retinol was applied showed a significant increase in TEWL compared to the other groups (Figure 25). On the other hand, in the group to which a substance that promotes the self-renewal ability of epidermal stem cells was applied, there was no difference in TEWL value from the control, suggesting that wrinkles are reduced while normal barrier function is maintained.

[0173] Since a significant increase in TEWL was observed with retinol, the skin flexibility (R0), which is said to reflect the state of the stratum corneum, was measured for mice in each group. As a result, the skin flexibility value was significantly reduced by UV exposure, but in the test group to which a substance that promotes the self-renewal ability of epidermal stem cells was applied, skin flexibility was significantly improved compared to the control group, retinol (Figure 26). This demonstrated that substances that promote the cell competitive ability of epidermal stem cells have the effect of improving wrinkles while maintaining good stratum corneum condition, compared to retinol.

[0174] Example 16: Skin roughness improvement test (apocynin) Using a rough skin model induced with a 10% sodium lauryl sulfate (SDS) solution (a standard test for skin damage caused by chemicals), a substance (apocynin) that promotes the competitive self-renewal ability of epidermal stem cells was administered to evaluate the effect of a rough skin prevention or improvement agent on improving rough skin.

[0175] Hairless mice HOS:HR-1 (female, 7 weeks old) were acclimatized to the experimental environment, and then divided into the following three groups so that the body weights were approximately uniform. SDS-untreated group (n=2) SDS treatment + solvent application group (n=4) SDS treatment + apocynin 200μM application group (n=4)

[0176] Cotton soaked in 500μL of 10% SDS solution (dissolved in 70% ethanol) was applied to the back of hairless mice for 30 minutes on the first, second, third, and fourth days. Apocynin was applied to the back every day from the day before the test to the fourth day. For mice in each group, TEWL was measured at specific sites on the back (two sites symmetrically on the left and right from the center of the spine) on the third day after the start of the test in the same manner as above. In addition, on the eighth day, images were taken with a high-resolution three-dimensional image analyzer PRIMOS-CR (Canfield Scientific, USA, Integral Corporation), and the degree of skin roughness was measured using Primos OMC3_22 software using parameters in the height direction that indicate the surface roughness of the skin, called "Ra (arithmetic mean roughness)" and "Rz (ten-point mean roughness)", at two sites symmetrically on the left and right from the center of the spine, the same as the sites where TEWL was measured.

[0177] The test results shown in Figures 27 and 28 show that in the SDS-treated control, TEWL increased significantly, and the Ra and Rz values, which are indicators of rough skin, increased, inducing significant rough skin. On the other hand, in mice that received a substance (apocynin) that promotes the self-renewal ability of epidermal stem cells, TEWL on the back was significantly decreased, and the Ra and Rz values ​​on the surface of the back were also significantly decreased, compared to the control group that received a solvent.

[0178] These results demonstrate that a substance that promotes the self-renewal ability of epidermal stem cells (apocynin) has a significant effect of preventing or improving rough skin.

[0179] Example 17: Skin roughness improvement test (ebselen) Using a rough skin model induced with a 10% sodium lauryl sulfate (SDS) solution, a substance that promotes the self-renewal ability of epidermal stem cells (ebselen) was administered to evaluate the effect of a rough skin prevention or improvement agent on improving rough skin.

[0180] Cotton soaked in 500μL of 10% SDS solution (dissolved in 70% ethanol) was applied to the back of hairless mice for 30 minutes on the first, second, third, and fourth days. Ebselen was applied to the back every day from the day before the test to the fourth day. For mice in each group, TEWL was measured at specific sites on the back (two sites symmetrically on the left and right from the center of the spine) on the eighth day after the start of the test in the same manner as above. At the same time, images were taken with a high-resolution three-dimensional image analyzer PRIMOS-CR (Canfield Scientific, USA, Integral Corporation), and the degree of skin roughness was measured using Primos OMC3_22 software with parameters in the height direction that represent surface roughness, called "Ra (arithmetic mean roughness)" and "Rz (ten-point mean roughness)", at two sites symmetrically on the left and right from the center of the spine, the same as the sites where TEWL was measured.

[0181] The test results shown in Figures 29 and 30 show that in the SDS-treated control, TEWL increased significantly, and the Ra and Rz values, which are indicators of rough skin, increased, inducing significant rough skin. On the other hand, in mice that received an application of a substance (ebselen) that promotes the self-renewal ability of epidermal stem cells, TEWL on the back was significantly decreased, and the Ra and Rz values ​​on the surface of the back were also significantly decreased, compared to the control group that received a solvent application.

[0182] These results demonstrate that a substance that promotes the self-renewal ability of epidermal stem cells (ebselen) has a significant effect in preventing or improving rough skin.

[0183] Example 18: Test to improve rough skin in humans Two subjects were given a skin roughness test induced by a 10% sodium lauryl sulfate (SDS) solution (a standard test for skin damage caused by chemicals in humans), and were administered substances that promote the self-renewal ability of epidermal stem cells (apocynin, ebselen, and celecoxib) to evaluate the effectiveness of skin roughness prevention or improvement agents in improving skin roughness.

[0184] Six square areas measuring 1.5 cm on each side were created on the inside of the subject's forearm, and the following treatments were performed on each area. ·SDS-untreated (70% ethanol-treated) group SDS treatment + solvent (50% ethanol / PBS) application group SDS treatment + apocynin (200μM) solution application group SDS treatment + Ebselen (0.25%) solution application group SDS treatment + celecoxib (2%) solution application group

[0185] Cotton soaked in 500μL of 10% SDS solution (dissolved in 70% ethanol) was applied to the test area for 30 minutes on days 0 and 1. Apocynin was applied to the test area before and after SDS treatment on day 0, and once a day until day 9. TEWL was measured for each test area before and after SDS treatment, and every day from days 1 to 7. The TEWL value on each site was calculated by subtracting the TEWL value before SDS treatment from the TEWL value on the measurement day, and this value was used as the evaluation value. In addition, the test area was photographed with a digital camera and a high-resolution three-dimensional image analyzer PRIMOS-CR (Canfield Scientific, USA, Integral Corporation). For the PRIMOS images, the degree of roughness was measured using the height parameters "Ry (maximum height)" and "Rz (ten-point average roughness)" that represent surface roughness, using Primos OMC3_22 software. For each site, the Rz or Ry value on the measurement day was subtracted from the Rz or Ry value of the non-SDS-treated control to obtain "ΔRz" and "ΔRy," and these values ​​were used for evaluation.

[0186] The test results are shown in Figures 31-33. As shown in Figure 31, redness and rough skin were clearly observed in the control and each solution-applied affected areas after SDS treatment, but on the 7th day, redness and rough skin continued in the affected areas where the control solution was applied, but were significantly alleviated in the areas where apocynin, ebselen, and celecoxib were applied. Furthermore, on the 16th day, pigmentation was observed in the affected areas of the control, but almost no pigmentation was observed in the affected areas where apocynin was applied. Also, as shown in Figure 32, the ΔTEWL value was significantly increased due to skin roughness induced by SDS, but the ΔTEWL value was significantly decreased by the application of apocynin, ebselen, and celecoxib. Furthermore, as shown in the results of Figure 33, when the skin texture was analyzed based on high-resolution photographs of the skin surface, it was found that rough skin was significantly induced in the SDS-treated control, but was significantly alleviated by the application of apocynin, ebselen, and celecoxib. Furthermore, the ΔRz and ΔRy values, which indicate the roughness of the skin surface and serve as indicators of skin roughness, increased significantly in the SDS-treated control, but these values ​​significantly decreased in the affected areas where apocynin, ebselen, and celecoxib were applied. This shows that the skin roughness and pigmentation induced significantly by the SDS solution can be significantly improved by substances that promote the competitive self-renewal ability of epidermal stem cells.

[0187] The results in Figures 31-33 indicate that substances that promote the competitive self-renewal ability of epidermal stem cells have a significant preventive or ameliorative effect on rough skin in humans, and are therefore considered to be effective in preventing and treating skin disorders caused by chemicals, etc.

[0188] Manufacturing Example 1 (1) Manufacturing of Apocynum venetum extract The rafuma extract used was made by Tokiwa Phytochemical Laboratory Co., Ltd. Dried leaves of rafuma (Apocynum venetum) were crushed, 60% ethanol was added to the crushed material, heated under reflux, and filtered to obtain an extract. The extraction residue was heated under reflux again in 60% ethanol, filtered, and obtained an extract. The first and second extracts were combined, concentrated under reduced pressure, the pH was adjusted to 3, and stirred overnight. Insoluble matter was removed by filtration, and the obtained filtrate was passed through a synthetic adsorption resin, washed with water, and the fraction desorbed with 70% ethanol was collected, concentrated under reduced pressure, and spray-dried to obtain the rafuma extract.

[0189] (2) Mulberry (Morus alba) extract The mulberry extract used was made by Tokiwa Phytochemical Laboratory Co., Ltd. 50% ethanol was added to mulberry (Morus alba) leaves, heated under reflux, and then filtered to obtain an extract. The extract was concentrated under reduced pressure, and then an excipient was added and dried to obtain the mulberry extract.

[0190] (3) Preparation of Gymnema sylvestre extract Gymnema extract was used from Tokiwa Phytochemical Laboratory Co., Ltd. Gymnema sylvestre leaves were crushed and extracted with 70% ethanol under heating and reflux, then filtered to obtain an extract. The extract was concentrated under reduced pressure and then dried under reduced pressure to obtain the Gymnema extract.

[0191] (4) Tea (Camellia sinensis) extract The tea extract used was manufactured by Tokiwa Phytochemical Laboratory Co., Ltd. Tea (Camellia sinensis) leaves were extracted with hot water and filtered to obtain an extract. The resulting extract was purified using an adsorption resin, concentrated under reduced pressure, and dried to obtain the tea extract.

[0192] (5) Milk thistle (Silybum marianum) extract The milk thistle extract used was made by Tokiwa Phytochemical Laboratory Co., Ltd. Milk thistle (Silybum marianum) fruit (defatted residue) was extracted by heating under reflux with 90-95% ethanol, and filtered to obtain an extract. The extract was concentrated under reduced pressure, purified with ethanol, concentrated under reduced pressure, and dried with hot air to obtain the milk thistle extract.

[0193] (6) Loquat (Eriobotrya japonica) Leaf Extract The loquat leaf extract used was manufactured by Tokiwa Phytochemical Laboratory Co., Ltd. Crushed loquat (Eriobotrya japonica) leaves were extracted with 80% ethanol under heating under reflux, and the extract was filtered to obtain an extract. Activated carbon was added to the extract to carry out a decolorization process, after which the decolorized solution was concentrated under reduced pressure and dried under reduced pressure to obtain a loquat leaf extract.

[0194] (7) Preparation of black turmeric (Kaempferia parviflora) extract The black turmeric extract used was that manufactured by Tokiwa Phytochemical Laboratory Co., Ltd. Dried chips of black turmeric (Kaempferia parviflora) rhizome were crushed in a mixer, 80% ethanol was added to the crushed material, heated under reflux, and filtered to obtain an extract. The extraction residue was heated under reflux again in 80% ethanol, and filtered to obtain an extract. The first and second extracts were combined and concentrated under reduced pressure, and then excipients were added and dried under reduced pressure to obtain a black turmeric extract.

[0195] Manufacturing Example 2 The plant extracts shown in Table 4 were extracted from each part of various plants using a designated extraction solvent.

[0196] [Table 4]

[0197] Although preferred embodiments of the present invention are shown herein, it will be apparent to those skilled in the art that such embodiments are provided merely for illustrative purposes, and that those skilled in the art will be able to make various modifications, changes, and substitutions without departing from the present invention. It should be understood that various alternative embodiments of the invention described herein may be used in carrying out the present invention. In addition, the contents of all publications, including patents and patent application documents referred to in this specification, should be construed as being incorporated by reference in the same manner as the contents set forth herein. This application claims priority to Japanese Patent Application No. 2019-59616 (filing date: March 27, 2019), which is incorporated herein in its entirety by reference. [Industrial Applicability]

[0198] As described above, the present inventors have found that COL17A1 (type XVII collagen) is involved in skin aging and regeneration, as well as skin damage caused by anticancer drugs and radiation, and have identified a composition that is useful for promoting skin wound healing, inhibiting skin aging, controlling cell competition, and improving the regenerative ability of epidermal stem cells. Continuing treatment due to skin damage caused by anticancer drugs has become a major issue, and is a problem related to prognosis. The number of cancer patients who continue working while continuing cancer treatment is increasing year by year, and the need for appearance care is extremely high. The composition, cells, and methods disclosed in the present application can be effectively used in the fields of continued anticancer drug treatment, surgical medicine, regenerative medicine, beauty, etc.

Claims

1. A composition for use in promoting skin wound healing or preventing or improving skin ulcers or bedsores, characterized in that it contains as an active ingredient a substance selected from the group consisting of substances that induce or maintain the expression of COL17A1 in cells, substances that suppress the degradation of COL17A1 in cells, substances that maintain the self-renewal ability of epidermal stem cells, substances that suppress genomic stress or oxidative stress in cells, and substances that suppress DNA damage in cells.

2. A composition for use in inhibiting skin aging, characterized in that it contains as an active ingredient a substance selected from the group consisting of substances that induce or maintain the expression of COL17A1 in cells, substances that inhibit the degradation of COL17A1 in cells, substances that maintain the self-renewal ability of epidermal stem cells, substances that inhibit genomic stress or oxidative stress in cells, and substances that inhibit DNA damage in cells.

3. A composition for enhancing the regenerative ability of epidermal stem cells, characterized in that it contains as an active ingredient a substance selected from the group consisting of a substance that induces or maintains the expression of COL17A1 in cells, a substance that inhibits the degradation of COL17A1 in cells, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that inhibits genomic stress or oxidative stress in cells, and a substance that inhibits DNA damage in cells.

4. A composition for use in preventing or improving skin damage caused by anticancer drugs, characterized in that it contains as an active ingredient a substance selected from the group consisting of substances that induce or maintain the expression of COL17A1 in cells, substances that suppress the degradation of COL17A1 in cells, substances that maintain the self-renewal ability of epidermal stem cells, substances that suppress genomic stress or oxidative stress in cells, and substances that suppress DNA damage in cells.

5. A composition for use in anti-wrinkle treatment, characterized in that it contains as an active ingredient a substance selected from the group consisting of substances that induce or maintain the expression of COL17A1 in cells, substances that suppress the degradation of COL17A1 in cells, substances that maintain the self-renewal ability of epidermal stem cells, substances that suppress genomic stress or oxidative stress in cells, and substances that suppress DNA damage in cells.

6. The composition according to claim 5, wherein the anti-wrinkle effect is achieved by at least one selected from the group consisting of improvement of skin elasticity, improvement of stratum corneum function, improvement of skin moisturizing ability, and improvement of skin barrier function.

7. A composition for use in anti-spot treatment, characterized in that it contains as an active ingredient a substance selected from the group consisting of substances that induce or maintain the expression of COL17A1 in cells, substances that suppress the degradation of COL17A1 in cells, substances that maintain the self-renewal ability of epidermal stem cells, substances that suppress genomic stress or oxidative stress in cells, and substances that suppress DNA damage in cells.

8. A composition for use in improving rough skin, characterized in that it contains as an active ingredient a substance selected from the group consisting of substances that induce or maintain the expression of COL17A1 in cells, substances that suppress the degradation of COL17A1 in cells, substances that maintain the self-renewal ability of epidermal stem cells, substances that suppress genomic stress or oxidative stress in cells, and substances that suppress DNA damage in cells.

9. The composition according to any one of claims 1 to 8, wherein the substance that induces or maintains the expression of COL17A1 in cells further maintains the self-renewal ability of epidermal stem cells.

10. The composition according to any one of claims 1 to 9, wherein the substance that induces or maintains expression of COL17A1 in a cell is selected from the group consisting of an NADPH oxidase inhibitor, a COX inhibitor, an iNOS inhibitor, a ROCK inhibitor and an estrogenic substance.

11. The composition of claim 10, wherein the NADPH oxidase inhibitor is selected from the group consisting of apocynin, ebselen, diphenyleneiodonium (DPI), GKT137831, AEBSF, GK-136901, ML171, Coenzyme Q10 (CoQ10), VAS2870 and VAS3947.

12. 11. The composition of claim 10, wherein the COX inhibitor is selected from the group consisting of celecoxib, deracoxib, tolfenamic acid, nifluric acid, FR122047, LM-1685, SC-791, BTB02472, nimesulide, SPB04674, curcumin, diclofenac, 4'-hydroxydiclofenac, DuP-697, ebselen, ETYA, flurbiprofen, ibuprofen, indomethacin, meloxicam, NPPB, NS-398, pterostilbene, resveratrol, SC-560, SKF-86002, TXA (tranexamic acid), TXC (tranexamic acid cetyl hydrochloride), astaxanthin, and sulindac sulfide.

13. 11. The composition of claim 10, wherein the iNOS inhibitor is selected from the group consisting of 1400W, L-NIL, aminoguanidine, BYK190123, S-ethylisothiourea, S-methylisothiourea, S-aminoethylisothiourea, 2-iminopiperidine, butylamine, ONO-1714 ((1S,5S,6R,7R)-7-chloro-3-imino-5-methyl-2-azabicyclo[4.1.0]heptane hydrochloride), AMT hydrochloride (2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine hydrochloride), AR-C102222, L-NG-nitroarginine, L-NG-monomethylarginine, L-nitroarginine methyl ester, L-NIO, dexamethasone, estrogen, astaxanthin, and apigenin.

14. The composition of claim 10, wherein the ROCK inhibitor is selected from the group consisting of Y-27632, Ripasudil (K-115), Thiazovivin, Fasudil (HA-1077), GSK429286A, RKI-1447, GSK269962, Netarsudil (AR-13324), Y-39983, ZINC00881524, KD025, Hydroxyfasudil (HA-1100), GSK180736A and AT13148.

15. 11. The composition of claim 10, wherein the estrogenic substance is selected from the group consisting of estrogen, ethinyl estradiol, biochanin A, soybean extract, isoflavones, Belamcanda chinensis extract, and Pueraria mirifica root extract.

16. The composition according to any one of claims 1 to 9, wherein the substance that induces or maintains expression of COL17A1 in a cell is selected from the group consisting of apocynin, ebselen, celecoxib, apigenin, Y-27632, ripasudil, 1400W, ethinyl estradiol, biochanin A, necrostatin 1, rafuma extract, mulberry extract, gymnema extract, tea extract, loquat leaf extract, milk thistle extract, black turmeric extract, psyllium oleracea seed extract, coffee seed extract, Somei-Yodoshino leaf extract, Melia azadirachta leaf extract, Mandarin orange peel extract, lavender flower extract, Sophora angustifolia root extract, rice bran extract, Rosa canina fruit extract, Hamemaris leaf extract, Eucalyptus leaf extract, Crataegus crenata extract, and carrot extract.

17. The composition according to any one of claims 1 to 9, wherein the substance that inhibits the degradation of COL17A1 in cells is selected from the group consisting of neutrophil elastase (ELANE) inhibitors and matrix metalloproteinase (MMP) inhibitors.

18. The composition of claim 17, wherein the ELANE inhibitor is selected from the group consisting of sivelestat sodium hydrate (Monosodium N-{2-[4-(2,2-dimethylpropanoyloxy)-phenylsulfonylamino]benzoyl}aminoacetate tetrahydrate), ONO-6818 (2-(5-Amino-6-oxo-2-phenylhydropyrimidinyl)-N-[2-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-1-(methylethyl)-2-oxoethyl]acetamide), alpha 1 antitrypsin (alpha 1-AT), Depelestat, Nilone (Sodium trifluoride isopropyl oxopropyl aminocarbonyl pyrrolidine carbonyl methylpropyl aminocarbonyl benzoyl aminoacetate), perilla leaf fermentation product, parsley fermentation product, bell pepper fermentation product, an antibody against ELANE, an siRNA against a gene encoding ELANE, and an antisense oligonucleotide against a gene encoding ELANE.

19. 18. The composition of claim 17, wherein the MMP inhibitor is selected from the group consisting of Marimastat, Batimastat, PD166793, Ro32-3555, WAY170523, UK370106, TIMP1, TIMP2, TIMP3, and TIMP4.

20. The composition according to any one of claims 1 to 9, wherein the genomic stress is any one of DNA damaging stress caused by ultraviolet light, radiation or an anticancer drug, and replication stress associated with cell division.

21. 3. The composition according to claim 2, wherein the skin aging is at least one selected from the group consisting of the following (a) to (f): (a) thinning, weakening, atrophy, or loss of firmness of the epidermis, dermis, and / or fat tissue (b) Decreased epidermal barrier function or dry skin (c) Reduction or immaturity of hemidesmosomes in the basement membrane (d) Loss of fibroblasts in the upper dermis or dry skin resulting in crinkled or fine wrinkles (e) Wrinkles due to loss of collagen in the dermis (f) Age spots, age spots, or depigmentation

22. A composition according to claim 2 for use in inhibiting wrinkles.

23. The composition according to any one of claims 1 to 22, which is a pharmaceutical composition.

24. The composition according to any one of claims 1 to 23, comprising a pharma- ceutically acceptable excipient.

25. The composition according to any one of claims 1 to 24, which is a coating agent.

26. The composition according to any one of claims 1 to 22, which is a cosmetic composition.

27. 27. The cosmetic composition of claim 26, comprising 0.01 to 15% by weight of the active ingredient.

28. 28. The cosmetic composition according to claim 26 or 27, which is a composition for use in skin care.

29. The cosmetic composition of any one of claims 26 to 28, further comprising at least one of a skin anti-aging agent, a skin toning agent, an anti-inflammatory agent, and a sunscreen agent.

30. A method for screening a substance effective in promoting skin wound healing or preventing or ameliorating skin ulcers, inhibiting skin aging, controlling cell competition, improving the regenerative ability of epidermal stem cells, preventing or ameliorating skin disorders caused by anticancer drugs, preventing wrinkles, and / or improving rough skin, comprising: i) contacting a cell with a test substance in vitro; ii) measuring the expression of COL17A1 in the cells; and iii) determining whether the test substance increases the expression of COL17A1. A method comprising:

31. A method for evaluating skin aging using the expression of COL17A1 in cells as an indicator.

32. A kit for use in a method for evaluating skin aging, comprising an antibody against COL17A1, or a probe for a gene encoding COL17A1, or a primer for amplifying the gene.

33. A method for evaluating epidermal stem cells using the expression of COL17A1 in the cells as an index.

34. A kit for use in a method for evaluating epidermal stem cells, comprising an antibody against COL17A1, or a probe for a gene encoding COL17A1, or a primer for amplifying said gene.

35. A method for selecting epidermal stem cells using the expression of COL17A1 in the cells as an indicator.

36. A kit for use in a method for selecting epidermal stem cells, comprising an antibody against COL17A1, or a probe for a gene encoding COL17A1, or a primer for amplifying the gene.

37. A method for expanding epidermal stem cells and / or epidermal cells, comprising the steps of measuring the expression level of COL17A1 using an antibody against COL17A1, or a probe for the gene encoding COL17A1, or a primer for amplifying the gene, and selecting epidermal stem cells having high expression levels of COL17A1.

38. A kit for use in a method for amplifying epidermal stem cells and / or epidermal cells, comprising an antibody against COL17A1, or a probe for a gene encoding COL17A1, or a primer for amplifying the gene.

39. A functional food or beauty supplement characterized by containing as an active ingredient a substance selected from the group consisting of substances that induce or maintain the expression of COL17A1 in cells, substances that inhibit the degradation of COL17A1 in cells, substances that maintain the self-renewal ability of epidermal stem cells, substances that inhibit genomic stress or oxidative stress in cells, and substances that inhibit DNA damage in cells.

40. 40. The functional food or beauty supplement of claim 39, wherein the substance that induces or maintains expression of COL17A1 in a cell is selected from the group consisting of NADPH oxidase inhibitors, COX inhibitors, iNOS inhibitors, ROCK inhibitors and estrogenic substances.

41. The functional food or beauty supplement according to claim 39 or 40, wherein the substance that induces or maintains the expression of COL17A1 in cells is selected from the group consisting of apocynin, ebselen, celecoxib, apigenin, Y-27632, ripasudil, 1400W, astaxanthin, ethinyl estradiol, biochanin A, necrostatin 1, rafuma extract, mulberry extract, gymnema extract, tea extract, loquat leaf extract, milk thistle extract, black turmeric extract, psyllium oleracea seed extract, coffee seed extract, Somei-Yodoshino leaf extract, Melia azadirachta leaf extract, Mandarin orange peel extract, lavender flower extract, Sophora angustifolia root extract, rice bran extract, Rosa canina fruit extract, Hamemaris leaf extract, Eucalyptus leaf extract, and Crataegus cratae extract.

42. The functional food or beauty supplement according to any one of claims 39 to 41, which is intended for oral intake.

43. 43. The functional food or cosmetic supplement of any one of claims 39 to 42, in the form of a tablet, powder, semi-solid, jelly or liquid.

44. 44. The functional food or beauty supplement of any one of claims 39 to 43, further comprising at least one of a skin anti-aging agent, a vitamin, collagen, and a mineral.

45. A cell composition comprising an isolated cell that expresses COL17A1.

46. The cell composition of claim 45, wherein the cells are epidermal keratinocytes or mucosal epithelial keratinocytes.

47. 47. A cell composition according to claim 45 or 46 for use in transplantation.

48. The cell composition of any one of claims 45 to 47 for use in treating a skin disease or injury.

49. At least 1 x 10 ３ The cell composition of any one of claims 45 to 48, comprising cells.

50. The cell composition of any one of claims 45 to 49, wherein at least 50% of the cells express COL17A1.

51. The cell composition of any one of claims 45 to 50, wherein the cells are cells derived from stem cells.

52. The cell composition of any one of claims 45 to 51, wherein the cells are frozen.

53. The cell composition of any one of claims 45 to 52, wherein the cells are contained in a single container.

54. The cell composition of any one of claims 45 to 53, wherein the cell is a cell that has been treated with a substance that induces or maintains expression of COL17A1 in the cell.

55. The cell composition of claim 53, wherein the substance that induces or maintains expression of COL17A1 in the cells is selected from the group consisting of NADPH oxidase inhibitors, COX inhibitors, iNOS inhibitors, ROCK inhibitors and estrogenic substances.

56. The cell composition of claim 54 or 55, wherein the substance that induces or maintains expression of COL17A1 in cells is selected from the group consisting of apocynin, ebselen, celecoxib, apigenin, Y-27632, ripasudil, 1400W, astaxanthin, ethinyl estradiol, biochanin A, necrostatin 1, rafuma extract, mulberry extract, gymnema extract, tea extract, loquat leaf extract, milk thistle extract, black turmeric extract, psyllium oleracea seed extract, coffee seed extract, Somei-Yodoshino leaf extract, Melia azadirachta leaf extract, Mandarin orange peel extract, lavender flower extract, Sophora angustifolia root extract, rice bran extract, Rosa canina fruit extract, Hamemaris leaf extract, Eucalyptus leaf extract, Crataegus crenata extract, and carrot extract.

57. A cell sheet comprising the cell composition according to any one of claims 45 to 56.

58. A composition for use in controlling cell competition, characterized in that it contains as an active ingredient a substance selected from the group consisting of a substance that induces or maintains the expression of COL17A1 in a cell, a substance that suppresses the degradation of COL17A1 in a cell, a substance that maintains the self-renewal ability of epidermal stem cells, a substance that suppresses genomic stress or oxidative stress in a cell, and a substance that suppresses DNA damage in a cell.

59. A method for evaluating cell competition in keratinocytes by culturing keratinocytes in three dimensions.

60. A kit for use in a method for evaluating cell competition in keratinocytes, comprising keratinocytes and a three-dimensional culture device.

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