Skin composition

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] This invention relates to compositions that have beneficial effects on the skin. More specifically, it relates to compositions that have the effect of promoting skin wound healing, inhibiting skin aging, and / or enhancing the regenerative capacity of epidermal stem cells. Furthermore, this invention relates more specifically to pharmaceutical compositions, cosmetic compositions, and beauty supplements. [Background technology]

[0002] The aging of animal organs is a multi-stage process that leads to their structural and functional decline. While cellular aging and / or stem cell depletion are related as cellular dynamics, the exact fate of depleted / stressed cells remains unclear. Generally, little is known about the in vivo dynamics of cells that make up aging tissues / organs. 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 miniaturization of hair follicles, leading to age-related hair thinning and graying (Non-patent Literature 1, Patent Literature 2). This raises a new fundamental question: do large solid organs such as skin have their own unique aging programs governing the cellular dynamics of organ aging?

[0003] Cell competition has been reported, primarily in Drosophila, to eliminate unsuitable mutant cells in epithelial tissue during embryonic development and cancer development. Stem cells are known to engage in "neutral stem cell competition" in some epithelial tissues, characterized by probabilistic loss and replacement of stem cells. However, it has not been clear whether this is a completely random selection of cell fate or a random occurrence of cell competition triggered by differences between cells. Furthermore, in the epidermis, symmetric cell division (SCD) and asymmetric cell division (ACD) of epidermal stem cells (keratinocyte stem cells, epidermal basal cells) are balanced to maintain epidermal homeostasis. However, the specific involvement and role of cell competition in maintaining physiological homeostasis and aging in mammalian organs, as well as in the cell dynamics of stratified epithelium, have remained unclear until now.

[0004] As human skin ages, it exhibits atrophy (thinning), fragility, dryness, hyperpigmentation, delayed wound healing, and increased irritation. During this process, the skin loses its epidermal and dermal thickness, epidermal ridges, epidermal keratinocytes, and a reserve of functional dermal fibroblasts. The fragility of aging human skin is partly attributable to the remodeling of the dermal-epidermal junction, particularly the 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) has been reported in aging human skin. 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, dyspigmentation (polymorphic cutaneous atrophy), and alopecia. Consistent with its phenotype, previous studies have shown that Col17a1 gene deficiency in mice causes follicular senescence with loss of hair follicle cells (Non-Patent Literature 2, Patent Literature 1). However, the relationship between the expression level of COL17A1 in epidermal stem cells of the skin and the expression of aging traits commonly seen in healthy human skin (skin atrophy, fragility, impaired barrier function, pigment abnormalities, hair loss, etc.) remains unclear. Furthermore, the involvement and role of COL17A1 expression in maintaining skin youthfulness, preventing aging, and promoting regeneration (wound healing), the correlation between COL17A1 expression levels and the competitive self-renewal capacity of stem cells, and whether stem cell competition by COL17A1 maintains the quality (youthfulness) of stem cells have not been clarified. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2009-161509 [Patent Document 2] International Open Pamphlet 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] [Problems to be Solved by the Invention]

[0007] One of the objectives of the present invention is to provide a method and a composition for preventing and improving skin ulcers, that is, for promoting skin wound healing. Another objective is to provide a method and a composition for reducing or preventing skin disorders associated with cancer treatment. Further, the present invention also aims to provide a method for suppressing skin aging, a composition for use in suppressing skin aging, and a composition for suppressing the aging of hair follicles, which are appendages of the skin. Additionally, the present invention aims 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 a method for controlling stem cell competition.

[0008] In addition, the present invention also aims to provide a method for screening substances effective in reducing or preventing skin disorders associated with cancer treatment, promoting skin wound healing, suppressing skin aging, controlling cell competition, and / or improving the regenerative ability of epidermal stem cells. [Means for Solving the Problems]

[0009] To elucidate the mechanisms of mammalian organ aging using mice with identical genetic and environmental backgrounds, we utilized the tail skin of C57BL6 inbred mice that exhibit age-related skin atrophy, fragility, pigmentation abnormalities, and impaired wound healing, similar to those seen in aged human skin. The inventors found that skin homeostasis is maintained by epidermal cell competition dynamics linked to COL17A1-mediated epidermal stem cell proliferation, thereby controlling skin organ aging. Furthermore, the inventors performed in vivo cell fate tracking analysis of epidermal stem cells in aging mice and found that COL17A1 in the epidermis high and COL17A1 low The heterogeneous emergence of clones promotes mutual cell competition among them through COL17A1-mediated SCD, and COL17A1 low COL17A1 linked to the elimination of clones high We revealed that it promotes the proliferation of epidermal stem cell clones. Furthermore, we showed that competitive dynamics of epidermal stem cells mediate the epidermal aging process, which involves a decrease in skin regeneration capacity and a reduction in epidermal melanocytes and dermal mesenchymal cells, thereby regulating the aging of the skin organ itself. In addition, we succeeded in preventing skin aging and improving 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 skin organs through competition among epidermal stem cells and the maintenance of heterogeneous cells.

[0010] Cell competition is associated with the elimination of unsuitable cells, but its role in the aging of mammalian organs has been largely unknown until now. The inventors have revealed that epidermal stem cells sense their cytocompatibility / stress through the stability of the HD component type XVII collagen (COL17A1) to promote cell competition for skin homeostasis and aging. In vivo cell fate tracking, clonal analysis, and in vitro three-dimensional modeling revealed that COL17A1 high Epidermal stem cells proliferate clonally via COL17A1-dependent symmetric cell division, thereby maintaining homeostasis by releasing COL17A1 from the skin.low / - It has been revealed that the exhausted cells are eliminated. Their dominant expansion by competition ultimately minimizes their own COL17A1 levels, not only reducing their self-renewal ability but also the renewal ability of adjacent epidermal melanocytes and dermal fibroblasts, causing skin atrophy, abnormal pigmentation, and poor wound healing. Furthermore, forced maintenance of COL17A1 expression in the epidermis rescues the phenotypic manifestations of skin aging. These findings open up new avenues for therapeutic intervention. The present invention is based on these findings and specifically relates to the following matters.

[0011] [Aspect 1] A composition for promoting skin wound healing, or preventing or improving skin ulcers or pressure ulcers, comprising 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 maintains the self-renewal ability of epidermal stem cells, a substance that suppresses genomic stress or oxidative stress in cells, and a substance that suppresses DNA damage in cells. [Aspect 2] A composition for suppressing skin aging, comprising 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 maintains the self-renewal ability of epidermal stem cells, a substance that suppresses genomic stress or oxidative stress in cells, and a substance that suppresses DNA damage in cells. [Aspect 3] A composition for enhancing the regenerative ability of epidermal stem cells, comprising 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 maintains the self-renewal ability of epidermal stem cells, a substance that suppresses genomic stress or oxidative stress in cells, and a substance that suppresses 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 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 5] A composition for anti-wrinkle use, 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 suppresses genomic stress or oxidative stress in cells, and a substance that suppresses DNA damage in cells. [Aspect 6] The composition according to embodiment 5, wherein the anti-wrinkle effect is due to 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. [Aspect 7] A composition for use in anti-blemish 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 suppresses genomic stress or oxidative stress in cells, and a substance that suppresses 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 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 suppresses genomic stress or oxidative stress in cells, and a substance that suppresses 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 a 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 estrogen-like substances. [Aspect 11] The composition according to 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] The composition according to embodiment 10, wherein the COX inhibitor is selected from the group consisting of celecoxib, delacoxib, tolfenamiic acid, difluic acid, FR122047, LM-1685, SC-791, BTB02472, nimeslide, SPB04674, curcumin, diclofenac, 4'-hydroxydiclofenac, DuP-697, ebselene, ETYA, fluviprofen, ibuprofen, indomethacin, meloxicam, NPPB, NS-398, pterostilbene, resveratrol, SC-560, SKF-86002, TXA (tranexamic acid), TXC (cetyl hydrochloride tranexamic acid), ginseng extract, and sulindac sulfide. [Aspect 13] The composition according to 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]heptanehydrochloride), AMT hydrochloride (2-amino-5,6-dihydro-6-methyl-4H-1,3-thiadin hydrochloride), AR-C102222, L-NG-nitroarginine, L-NG-monomethylarginine, L-nitroarginine methyl ester, L-NIO, dexamethasone, estrogen, astaxanthin, and apigenin. [Aspect 14] The composition according to 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, netalusdil (AR-13324), Y-39983, ZINC00881524, KD025, hydroxyfasudil (HA-1100), GSK180736A, and AT13148. [Aspect 15] The composition according to embodiment 10, wherein the estrogen-like substance is selected from the group consisting of estrogen, ethinylestradiol, biochanin A, soybean extract, isoflavone, Belamcanda chinensis extract, and Pueraria mirifica root extract. [Aspect 16] A composition according to any one of embodiments 1 to 9, wherein a substance that induces or maintains the expression of COL17A1 in cells is selected from the group consisting of apocynin, ebselen, celecoxib, apigenin, Y-27632, lipasudil, 1400W, ethinylestradiol, biochanin A, necrostatin 1, Rhodiola rosea extract, Mulberry extract, Gymnema sylvestris extract, Tea extract, Loquat leaf extract, Milk thistle extract, Black turmeric extract, Plantago major seed extract, Coffee seed extract, Prunus yedoensis leaf extract, Melia azadirachta leaf extract, Mandarin orange peel extract, Lavandula angustifolia flower extract, Sophora flavescens root extract, Rice bran extract, Rosa canina fruit extract, Haemaphyllosa leaf extract, Eucalyptus globulus leaf extract, Crataegus monogyna extract, and Carrot extract. [Aspect 17] The composition according to any one of embodiments 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. [Aspect 18] ELANE inhibitors include sivelestat sodium hydrate (Monosodium N-{2-[4-(2,2-dimethylpropanoyloxy)-phenylsulfonylamino]benzoyl}aminoacetate) A composition according to embodiment 17, selected from the group consisting of 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), α1-antitrypsin (α1-AT), Depelestat, Neil One (sodium trifluoride isopropyl oxopropylaminocarbonylpyrrolidinecarbonylmethylpropylaminocarbonylbenzoylaminoacetate), perilla leaf ferment, parsley ferment, bell pepper ferment, antibody against ELANE, siRNA against the gene encoding ELANE, and antisense oligonucleotide against the gene encoding ELANE. [Aspect 19] The composition according to embodiment 17, wherein the MMP inhibitor is selected from the group consisting of Marimast, Batimastat, PD166793, Ro32-3555, WAY170523, UK370106, TIMP1, TIMP2, TIMP3, and TIMP4. [Aspect 20] The composition according to any one of embodiments 1 to 9, wherein the genomic stress is DNA damage stress caused by ultraviolet light, radiation, or anticancer drugs, or replication stress associated with cell division. [Aspect 21] The composition according to embodiment 2, wherein skin aging is at least one selected from the group consisting of (a) to (f) below. (a) Thinning, weakening, atrophy, or loss of firmness of the epidermis, dermis, and / or adipose tissue (b) Impaired 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 fine wrinkles or crepe wrinkles due to dry skin. (e) Wrinkle formation due to a decrease in collagen in the dermis (f) age spots, lentigines, or depigmented spots [Aspect 22] The composition according to embodiment 2 for use in suppressing wrinkles. [Aspect 23] A pharmaceutical composition, as described in any one of embodiments 1 to 22. [Aspect 24] A composition according to any one of embodiments 1 to 23, comprising a pharmaceutically acceptable excipient. [Pattern 25] A composition according to any one of embodiments 1 to 24, which is a coating agent. [Aspect 26] A cosmetic composition, as described in any one of embodiments 1 to 22. [Aspect 27] A cosmetic composition according to embodiment 26, containing an active ingredient in an amount of 0.01 to 15% by weight. [Aspect 28] A cosmetic composition according to embodiment 26 or 27, which is a composition for use in skincare. [Aspect 29] A cosmetic composition according to any one of embodiments 26 to 28, further comprising at least one of a skin anti-aging agent, a skin tone adjuster, an anti-inflammatory agent, and a sunscreen. [Aspect 30] A method for screening substances that are effective in promoting the healing of skin wounds or preventing or improving skin ulcers, inhibiting skin aging, controlling cell competition, improving the regenerative capacity of epidermal stem cells, preventing or improving skin damage caused by anticancer drugs, anti-wrinkle, and / or improving rough skin, i) A step of bringing cells and the test substance into contact in vitro. ii) A step of measuring the expression of COL17A1 in cells, and iii) A step of determining whether the test substance increases the expression of COL17A1. Methods that include... [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 of evaluating skin aging, comprising an antibody against COL17A1, or a probe for the gene encoding COL17A1, or a primer for amplifying said gene. [Aspect 33] A method for evaluating epidermal stem cells using COL17A1 expression in cells as an indicator. [Aspect 34] A kit for use in evaluating epidermal stem cells, comprising an antibody against COL17A1, or a probe for the gene encoding COL17A1, or a primer for amplifying said gene. [Aspect 35] A method for selecting epidermal stem cells using COL17A1 expression in 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 the gene encoding COL17A1, or a primer for amplifying said gene. [Aspect 37] A method for amplifying epidermal stem cells and / or epidermal cells, comprising the step 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 with high expression levels of COL17A1. [Aspect 38] A kit for use in a method of amplifying epidermal stem cells and / or epidermal cells, comprising an antibody against COL17A1, or a probe for the gene encoding COL17A1, or a primer for amplifying said 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 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 40] A functional food or beauty 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 estrogen-like substances. [Aspect 41] A functional food or beauty supplement according to embodiment 39 or 40, wherein a substance that induces or maintains the expression of COL17A1 in cells is selected from the group consisting of apocynin, ebselen, celecoxib, apigenin, Y-27632, lipasudil, 1400W, ethinylestradiol, biochanin A, necrostatin 1, Rhodiola rosea extract, Mulberry extract, Gymnema sylvestris extract, Tea extract, Loquat leaf extract, Milk thistle extract, Black turmeric extract, Plantago major seed extract, Coffee seed extract, Prunus yedoensis leaf extract, Melia azadirachta leaf extract, Mandarin orange peel extract, Lavandula angustifolia flower extract, Sophora flavescens root extract, Rice bran extract, Rosa canina fruit extract, Hamemaris radiata leaf extract, Eucalyptus globulus leaf extract, Hawthorn extract, and Carrot extract. [Aspect 42] A functional food or beauty supplement according to any one of descriptions 39 to 41, intended for oral intake. [Aspect 43] A functional food or beauty supplement according to any one of embodiments 39 to 42, which is in the form of a tablet, powder, semi-solid, jelly, or liquid. [Aspect 44] A functional food or beauty supplement according to any one of embodiments 39 to 43, further comprising at least one of a skin anti-aging agent, a vitamin, collagen, and a mineral. [Aspect 45] A cell composition comprising isolated cells expressing COL17A1. [Aspect 46] The cell composition according to embodiment 45, wherein the cells are epidermal keratinocytes or mucosal epithelial keratinocytes. [Aspect 47] A cell composition according to embodiment 45 or 46 for use in transplantation. [Aspect 48] A cell composition according to any one of embodiments 45 to 47, for use in the treatment of skin diseases or injuries. [Aspect 49] at least 1 × 10 3 A cell composition according to any one of embodiments 45 to 48, comprising a single cell. [Aspect 50] A cell composition according to any one of embodiments 45 to 49, wherein at least 50% of the cells express COL17A1. [Aspect 51] A cell composition according to any one of embodiments 45 to 50, wherein the cells are stem cell-derived cells. [Aspect 52] A cell composition according to any one of embodiments 45 to 51, wherein the cells are frozen. [Aspect 53] A cell composition according to any one of embodiments 45 to 52, wherein the cells are contained in a single container. [Aspect 54] A cell composition according to any one of embodiments 45 to 53, wherein the cells are cells treated with a substance that induces or maintains the expression of COL17A1 in the cells. [Aspect 55] The cell composition according to embodiment 53, wherein a 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 estrogen-like substances. [Aspect 56] A cell composition according to embodiment 54 or 55, wherein a substance that induces or maintains the expression of COL17A1 in cells is selected from the group consisting of apocynin, ebselen, celecoxib, apigenin, Y-27632, lipasudil, 1400W, ethinylestradiol, biochanin A, necrostatin 1, Rhodiola rosea extract, Mulberry extract, Gymnema sylvestris extract, Tea extract, Loquat leaf extract, Milk thistle extract, Black turmeric extract, Plantago major seed extract, Coffee seed extract, Prunus yedoensis leaf extract, Melia azadirachta leaf extract, Mandarin orange peel extract, Lavandula angustifolia flower extract, Sophora flavescens root extract, Rice bran extract, Rosa canina fruit extract, Hamemaris radiata leaf extract, Eucalyptus globulus leaf extract, Hawthorn extract, and Carrot extract. [Aspect 57] A cell sheet comprising the cell composition described in 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 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 suppresses genomic stress or oxidative stress in cells, and a substance that suppresses DNA damage in cells. [Aspect 59] A method for evaluating cell competition in keratinocytes by culturing them in three dimensions. [Aspect 60] A kit for evaluating cell competition in keratinocytes, including keratinocytes and a 3D culture device. [Brief explanation of the drawing]

[0012] [Figure 1] This graph quantifies the fluorescence intensity of HD and basement membrane components, COL17A1, ITGA6, PLECTIN, ITGB4, COL7A1, and ITGB1 in the tail of young (7-8 weeks old, n=50) and aged (22-25 months old, n=50) mice. COL17A1 and PLECTIN expression are significantly reduced in aged skin. [Figure 2] This graph quantifies the number of hemoglobin (HDs) in the basement membrane of tail basal cells from young (8 weeks old, n=3) and aged (22 months old, n=3) mice. Physiological aging significantly reduces the number of HDs in tail basal cells. [Figure 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 images of COL17A1 in multicolor-labeled tail basal keratinocytes from young (11 weeks old) and aged (28 months old) tamoxifen (TAM) treated Epi-Confetti mice. [Figure 5] Graphs quantifying the distribution between COL17A1 intensity and keratinocyte clonal size in multiple regions (11 weeks old, n=13 clones; 28 months old, n=12 clones) shown in Figure 4. [Figure 6] Graphs quantifying suspension clones in control (Cont) mice (D2, n=4; D28, n=5) and Col17a1 cKO mice (D2, n=3; D28, n=5) on day 2 (D2) and day 28 (D28) after TAM administration. [Figure 7] This graph analyzes colony formation of tail epidermal keratinocytes in young mice (7 weeks old, n=3) and aged mice (25 months old, n=3). The number and size of colonies significantly decrease with age. [Figure 8]This graph analyzes colony formation of tail epidermal keratinocytes in 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] This graph shows the changes in pigment cells associated with aging. As we age, pigment cells in the epidermis disappear, leading to skin pigment abnormalities (a mixture of deposition and desorption) (left, center). However, the forced maintenance of COL17A1 helps maintain pigment cells, thus suppressing these pigment abnormalities (right). [Figure 10] Graph showing the number of PDGFRa+ cells in wild-type and hCOL17A1tg. The graph illustrates the decrease in PDGFRa+ cells directly beneath the basement membrane (BM) due to aging (left) and its suppression by the forced maintenance of COL17A1 (right). [Figure 11] Graphs quantifying the unhealed wound area on day 0 (D0), day 14 (D14), and day 21 (D21). Physiological aging (left) or Col17a1 or Itga6 deficiency (right) significantly slows wound healing. [Figure 12] Graphs quantifying the unhealed wound area on day 0 (D0), day 14 (D14), and day 21 (D21). hCOL17A1 expression (left) and administration of apocynin or Y-27632 (right) significantly promoted wound repair. [Figure 13] Graph showing the results of the 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 greater than 2 mm (n=3 wells) was significantly increased by treatment with either Y-27632 or apocynin (right). [Figure 14] Graph showing the results of the 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 showing HCS analysis of COL17A1 expression (corresponding to Step 1 of screening for competitive self-renewal promoters mediated by COL17A1). In HCS, treatment with apocynin or ebselen significantly increased COL17A1 levels compared to the control. [Figure 16] Photographs and graphs analyzing the results of a colony formation assay (equivalent to Step 2 of the screening of competitive self-renewal promoters mediated by COL17A1 expression). Treatment with apocynin or ebselen significantly increased the number and area of ​​colonies per well compared to the control. [Figure 17] Decreased COL17A1 expression due to anticancer drugs and various stresses. Top image: Immunostaining image of COL17A1 expression in the skin of C57BL / 6 mice 3 months after continuous administration of hydroxyurea (HU). Middle left image: Western blotting image of COL17A1 levels 24 hours after irradiation of HaCaT cells with ultraviolet light. Middle image: Western blotting image of COL17A1 levels 72 hours after irradiation of HaCaT cells with radiation. Middle right image: Western blotting image of COL17A1 levels 24 hours after treatment of HaCaT cells with hydrogen peroxide. Bottom image: Western blotting image of COL17A1 levels when HaCaT cells were treated with an EGF receptor inhibitor (PD168393). [Figure 18] A graph showing the improvement effect of COL17A1 overexpression on skin disorders caused by anticancer drugs. [Figure 19] Graphs showing the improvement effect of competitive epidermal stem cell amplification agents on skin damage caused by anticancer drug administration. Left graph: Graph showing TEWL on day 12 after continuous administration of an EGF receptor inhibitor (erlotinib) to 7-week-old C57BL / 6 mice. Right graph: Graph showing TEWL on day 7 after continuous administration of an EGF receptor inhibitor (erlotinib) to 6-month-old C57BL6 mice. [Figure 20] This figure shows the preventive and therapeutic effects of apocynin in a model of skin disorders (dry eczema and alopecia) induced in the upper dorsal skin of the neck of aged mice. [Figure 21]Bar graphs showing the effects of substances that promote the competitive ability of epidermal stem cells on retaining moisture in the stratum corneum in response to UV irradiation (top) and suppressing the increase in TEWL (bottom). [Figure 22] The bar graph (top) shows the inhibitory effect of a competitive amplifier for epidermal stem cells on the increase in TEWL caused by UV irradiation, and the bar graph (bottom) shows the effect on improving decreased skin elasticity. [Figure 23] The graph (top) shows the effect of long-term continuous application of a competitive amplifier of epidermal stem cells and a comparator on improving skin elasticity. The bar graph (bottom) shows the TEWL (Thoroughly Activated Water Loss) when a competitive amplifier of epidermal stem cells and a comparator are applied long-term. Retinol, known as a wrinkle-reducing agent, significantly increases TEWL with long-term application. On the other hand, the TEWL value of substances that promote the competitive ability of epidermal stem cells is not significantly different from the control, suggesting that they improve skin elasticity while maintaining the skin's barrier function. [Figure 24] The image shows high-resolution photographs and colored images of wrinkles (top) demonstrating the improvement effect of a competitive amplification agent and a comparator on epidermal stem cells on wrinkles induced by UV irradiation. Also shown are bar graphs illustrating the total wrinkle volume (mm³) and the average total wrinkle depth (μm). [Figure 25] This bar graph shows the TEWL (Tissue-to-Wrinkle) levels after long-term application of competitive amplifiers for epidermal stem cells. Retinol, known as a wrinkle-reducing agent, significantly increases TEWL with long-term application. On the other hand, the TEWL values ​​of substances that promote the competitive ability of epidermal stem cells are not significantly different from the control, suggesting that they improve wrinkles while maintaining the skin's barrier function. [Figure 26] A bar graph showing the improvement in reduced skin elasticity caused by UV irradiation by a substance that promotes the competitive ability of epidermal stem cells and a comparator. [Figure 27] High-resolution photographs and colored images of skin texture showing the improvement effect of SDS-induced skin roughness by a competitive amplifier for epidermal stem cells (apocynin). The graphs show "Ra (arithmetic mean roughness)" and "Rz (ten-point mean roughness)," which are indicators of skin surface roughness. [Figure 28]This bar graph shows the effect of apocynin, a substance that promotes the cell competitiveness of epidermal stem cells, on suppressing the enhancement of TEWL induced by SDS. [Figure 29] High-resolution photographs and colored images of skin texture showing the improvement of SDS-induced skin roughness by a competitive amplification agent (ebselen) for epidermal stem cells. The graphs show "Ra (arithmetic mean roughness)" and "Rz (ten-point mean roughness)," which are indicators of skin surface roughness. [Figure 30] This bar graph shows the effect of ebselen, a substance that promotes the cell competitiveness of epidermal stem cells, on suppressing the enhancement of TEWL induced by SDS. [Figure 31] Photographs showing the effects of competitive epidermal stem cell amplifiers (apocynin, ebselen, celecoxib) on primary irritant skin damage (skin roughness) and hyperpigmentation induced by SDS. SDS treatment caused severe redness and dryness, which were significantly reduced by the application of the competitive epidermal stem cell amplifiers. At 16 days after the start of SDS treatment, the apocynin-treated area showed healing, while hyperpigmentation occurred in the control group. [Figure 32] High-resolution photographs and colored images of skin texture showing the improvement effect of competitive epidermal stem cell amplifiers (apocynin, ebselen, celecoxib) on SDS-induced skin roughness in humans. The graphs show "ΔRz" and "ΔRy" as the values ​​obtained by subtracting "Ry (maximum height)" and "Rz (ten-point mean roughness)," which are indicators of skin surface roughness, from the control value on the measurement day. [Figure 33] This graph shows the effect of competitive epidermal stem cell amplifiers (apocynin, ebselen, celecoxib) on suppressing the increase in TEWL associated with SDS-induced human skin irritation. The error bars in the graph indicate standard deviation (SD). [Modes for carrying out the invention]

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

[0014] skin wound healing Skin wounds can occur for various reasons, including trauma, cuts, burns, poor blood circulation, bedsores, and diabetes, temporarily impairing normal function and structure of the skin. If the body is unable to heal these wounds, they become chronic and eventually become infected. Skin wounds are classified into acute and chronic skin wounds. Acute skin wounds are those in which the wound healing mechanism functions normally, such as fresh trauma or surgical wounds, while chronic skin wounds are those in which the normal wound healing mechanism does not function 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. One percent of patients develop chronic skin wounds, but half of these wounds never heal completely.

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

[0016] The epidermis is further classified into four layers from the deepest to the deepest: the basal layer (layer of basal cells), the spinous layer (layer of spinous cells), the granular layer (layer of granular cells), and the stratum corneum (layer of keratinocytes). The basal layer consists of a single layer of basal cells containing keratinocyte stem cells. The basal layer has desmosomes, gap junctions, and hemidesmosomes as structures for connecting 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, hence they are called spinous cells. The granular layer consists of 2 to 3 layers. It is so named because it contains basophilic keratohyalin granules rich in profilaggrin. The stratum corneum, also called the stratum corneum, consists of about 10 layers. Denucleated and dead keratinocytes become membranous, forming a layered structure like a bed of fallen leaves. The stratum corneum is extremely thick in the palms and soles of the feet, with a layer of stratum lucidum directly beneath it. The cytoplasm of keratinocytes is filled with aggregated keratin fibers. Furthermore, hair follicles and sweat glands are present continuously with the epidermis, acting as skin appendages that produce hair and release sweat.

[0017] Epidermal stem cells, located in the basal layer of the epidermis, are the driving force behind epidermal regeneration. They perform balanced divisions, either unidirectional (horizontal division) or unequal (vertical division) relative to the basement membrane. They maintain the structure of the epidermis by supplying differentiated epidermal keratinocytes toward the skin surface while self-replicating. Keratinocytes make up the majority of cells in the epidermis. Almost all cells found in the stratum corneum of the skin surface are dead cells. When these dead cells slough off, new keratinocytes rise from the lower layers and replace them. This process of cells rising from below is constantly repeated, and the entire skin surface is renewed every 15 to 30 days.

[0018] The basement membrane lies beneath the epidermis, separating it from the dermis. The dermis is composed 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 it, and fibroblasts produce collagen protein for strength and elastin protein for elasticity.

[0019] At the deepest layer of the skin is the subcutaneous tissue. The subcutaneous tissue is a layer of fat beneath the dermis, and it acts as another barrier that absorbs shock and protects the muscles and bones below from infection.

[0020] When the epidermis layer of the skin is damaged by trauma, infection, anticancer drugs, age-related skin atrophy, breakdown of barrier function, or other skin diseases, a wound is formed, and the severity of the wound increases when damage occurs to the lower layers of the skin. Three steps are necessary for a wound to heal properly: inflammation, proliferation, and remodeling. Inflammation lasts for about four days. The main thing that happens during this period is the formation of fibrin matrix and the formation of a blood clot to cover and protect the wound. During the proliferation phase, inflammatory signals attract many types of cells to the wound site. These cells include fibroblasts and vascular endothelial cells. Fibroblasts produce collagen and can also transform into myofibroblasts to close the wound. Vascular endothelial cells create new blood vessels around the wound. Through the activity of these cells, a tissue called "granulation tissue" is formed just below the blood clot. Granulation tissue is new tissue that is formed as a repair-inflammatory response to tissue damage, and it is composed of new blood vessels, connective tissue, fibroblasts, and inflammatory cells. Next, epidermal regeneration occurs from the surrounding epidermis and skin appendages, with keratinocytes migrating from the wound edge to the wound site and also from the hair follicle base. In deep skin defects where no appendages remain, the epidermis extends from the surrounding area after the wound surface has been replaced by granulation tissue. Keratinocytes divide on the granulation tissue, bringing the epidermis together. This process continues throughout the remodeling stage, with epidermal keratinocytes migrating from the periphery of the wound or remaining appendages, restoring the epidermis. Keratinocytes and fibroblasts produce matrix proteins for the newly formed basement membrane, regenerating the interface between the epidermis and dermis.

[0021] In cases of severe deep burns or cuts, where damage extends from the dermis to the adipose tissue, hair follicles and sweat glands may also be destroyed, reducing the number of keratinocytes available for epidermal repair. As a result, full-thickness wounds take a long time to heal, and treatment using cultured skin such as skin grafts or epidermal sheets, or epidermal keratinocytes via spray transplantation may be necessary.

[0022] A composition for use in promoting the healing of skin wounds, or in preventing or improving skin ulcers or pressure ulcers. One embodiment of the present invention relates to a composition for use in promoting the healing of skin wounds or in preventing or improving skin ulcers or pressure ulcers, characterized in that it contains as an active ingredient 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 stress or oxidative stress in cells, and a substance that inhibits DNA damage (DNA damage response or DNA damage itself) in cells (epidermal stem cells).

[0023] Substances that induce or maintain COL17A1 expression in cells As described above, the inventors have successfully prevented skin aging, improved or accelerated wound healing, and prevented or improved skin ulcers or pressure ulcers by forcibly maintaining the expression of COL17A1 in epidermal stem cells, demonstrating that COL17A1 regulates skin organ aging through competition among epidermal stem cells and maintenance of heterogeneous cells. The inventors have also revealed that the matrix metalloproteinase (MMP) inhibitor marimasut stabilizes COL17A1 and blocks UV-induced downsuppression of COL17A1. Furthermore, the inventors have identified ROCK inhibitor (Y27632) and NADPH oxidase inhibitor (apocynin) as chemical substances that maintain or induce COL17A1 expression in keratinocytes in vitro and maintain and promote self-renewal ability in cultured keratinocytes.

[0024] Furthermore, the inventors have shown, both Y27632 (ROCK inhibitor) and apocynin (NADPH oxidase inhibitor) significantly promote the wound repair process, similar to transgenic mice overexpressing human COL17A1, both in vitro and in vivo. Therefore, it will be understood by those skilled in the art that substances that induce or maintain the expression of COL17A1 in cells, or substances that inhibit the degradation of COL17A1 in cells, can be used to promote or improve skin wound healing, or to prevent or improve skin ulcers or pressure ulcers. Furthermore, it will be understood that, in the present invention, substances that maintain the self-renewal capacity of epidermal stem cells, substances that suppress genomic stress or oxidative stress in cells, and substances that suppress DNA damage in cells can be used to promote or improve skin wound healing, or to prevent or improve skin ulcers or pressure ulcers.

[0025] COL17A1 (type XVII collagen / BP180 / BPAG2) is a transmembrane protein cell adhesion molecule and a protein of hemidesmosomes (semi-adhesion plaques), which are a type of cell adhesion in epithelial tissue. 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. Substances that induce or maintain COL17A1 expression in cells also include DNA or RNA encoding COL17A1. DNA or RNA encoding 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, netalusdil (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 such as Rhodiola rosea extract containing apocynins are also included.

[0027] Of the plant extracts used in this invention, the following are manufactured by Tokiwa Botanical Chemical Research Institute Co., Ltd. and are available for purchase: Rhodiola rosea extract (product name: Benetron), Mulberry extract (product name: Mulberry leaf extract powder), Gymnema sylvestris extract (product name: Gymnema extract powder), Tea extract (product name: Tiacaron 90), Loquat leaf extract (product name: Loquat leaf extract powder), Milk thistle extract (product name: Milk thistle extract powder), and Black turmeric extract (product name: Sartmax). Plantago major seed extract (product name: Absolage), coffee seed extract (product name: Café 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 Clear), lavender flower extract (product name: Eco Farm Lavender B), Sophora flavescens root extract (product name: Falcorex Sophora flavescens B), rice bran extract (product name: Falcorex Rice Bran BK), Rosa canina extract (product name: Falcorex Rosehip 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 Falcos Co., Ltd. product guide and are readily available.

[0028] Both selective and non-selective iNOS inhibitors can be used, but selective iNOS inhibitors are preferred, for example, from the standpoint of side effects. Examples of iNOS inhibitors include 1400W, L-NIL, aminoguanidine, BYK190123, S-ethylisothiourea, S-methylisothiourea, S-aminoethylisothiourea, 2-iminopiperidine, butylamine, and ONO-1714 (fusion piperidine derivative; (1S,5S,6R,7R)-7-chloro-3-imino-5-methyl-2-azabicyclo[4.1.0]heptanehydr). Examples include, but are not limited to, locloroxide, 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, delacoxib, tolfenamiic acid, FR122047, LM-1685, SC-791, BTB02472, nimeslid, SPB04674, curcumin, diclofenac, 4'-hydroxydiclofenac, DuP-697, ebselen, ETYA, fluviprofen, ibuprofen, indomethacin, meloxicam, difluic acid, NPPB, NS-398, pterostilbene, resveratrol, SC-560, SKF-86002, TXA (tranexamic acid), TXC (cetyl tranexamate hydrochloride), ginseng extract, astaxanthin, and sulindac sulfide. Both selective and non-selective COX inhibitors can be used, but selective COX-2 inhibitors are preferred, for example, from the standpoint of side effects.

[0030] Examples of estrogen-like substances include, but are not limited to, estrogen, ethinylestradiol, biochanin A, soybean extract, isoflavones, iris extract, and Pueraria mirifica root extract.

[0031] Substances that inhibit the degradation of COL17A1 in cells Substances that inhibit the degradation of COL17A1 in cells include, for example, neutrophil elastase (ELANE) inhibitors and MMP inhibitors. Examples of ELANE inhibitors include sibelestat 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, those containing -hNE4 or DX-890, Neal One (sodium trifluoride isopropyl oxopropylaminocarbonylpyrrolidinecarbonylmethylpropylaminocarbonylbenzoylaminoacetate), 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] ELANE inhibitors may include antibodies against ELANE, siRNA against genes encoding ELANE, or antisense oligonucleotides against genes encoding ELANE. Examples of MMP inhibitors include, but are not limited to, Marimast, Batimastat, PD166793, Ro32-3555, WAY170523, UK370106, TIMP1, TIMP2, TIMP3, and TIMP4.

[0033] Substances that maintain the self-renewal ability of epidermal stem cells The 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. Examples include apocynin, ebselen, celecoxib, apigenin, Y-27632, lipasudil, 1400W, astaxanthin, ethinylestradiol, biochanin A, necrostatin 1, rhodiola extract, mulberry extract, gymnema extract, tea extract, loquat leaf extract, milk thistle extract, black turmeric extract, plantain seed extract, coffee seed extract, cherry blossom leaf extract, melia azadirachta leaf extract, mandarin orange peel extract, lavender flower extract, Sophora flavescens root extract, rice bran extract, rosehip fruit extract, Hamemaris leaf extract, eucalyptus leaf extract, hawthorn extract, and carrot extract.

[0034] These substances maintain or promote the self-renewal ability of epidermal stem cells. In vivo, or in 3D cultured epidermis, epidermal stem cells expressing sufficient amounts of COL17A1 compete with surrounding epidermal stem cells that express low amounts of COL17A1, occupying and amplifying within the epidermis. Therefore, the self-renewal ability based on COL17A1 expression 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 substances mentioned above can also be called "competitive amplifiers for epidermal stem cells."

[0035] Substances that suppress genomic stress or oxidative stress in cells Furthermore, the inventors have found that the expression of COL17A1 decreases under various stresses. In this invention, the stresses to be suppressed are genomic stress (genotoxic stress) and oxidative stress. 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. Cellular function is impaired by stress that damages DNA or by blocking self-renewal signals caused by molecularly targeted drugs. Therefore, those skilled in the art will understand that skin wound healing can be promoted and improved by using substances that suppress genomic stress or oxidative stress in cells, or substances that suppress DNA damage in cells.

[0036] Substances that suppress DNA damage in cells Substances that suppress DNA damage include, for example, extracts of Sarcandra glabra, Saraca dives, Cudrania pubescens, Taxodium distichum, Ludwigia octovalis, Deutzianthus tonkinensis, Alchornea trewioides, Berchemia polyphylla, Glochidion puberum, and Sassafras. Extracts such as cinchona extract (see Japanese Patent Publication No. 2008-247854), cinchona extract, comfrey extract, coffee tree extract, kudzu extract, burdock extract, Coptis japonica extract, Sophora flavescens extract, Chlorella extract, lavender extract, evening primrose extract, rose extract, Gynostemma pentaphyllum extract, Enmeisou extract, Lamium album extract, carrot extract, linden extract, citronella extract, lotus extract, tea extract, or okra extract (see WO2013 / 031003).

[0037] The DNA damage inhibitory 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 focus induced by the DNA damage response. A substance that promotes DNA damage repair may also be used as the substance that suppresses DNA damage. Examples of agents that promote DNA damage repair include Gigartina tenella extract (see Japanese Patent Application Publication No. 2006-273761) or cinchona extract (see WO2013 / 031003). 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, UV absorbers, UV scatterers, radiation protectants, 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 suppressing skin aging, characterized in that it 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. One embodiment of the present invention may be a composition used particularly for inhibiting epidermal aging. Some embodiments of the present invention may be a composition used particularly for inhibiting skin aging, including hair follicles. Other embodiments of the present invention may be a composition used particularly for inhibiting skin aging, excluding hair follicles.

[0039] Furthermore, compositions used to suppress skin aging contain the active ingredients described in the section "Compositions used to promote the healing of skin wounds, or to prevent or improve skin ulcers or pressure ulcers," and in this specification, the active ingredients of the compositions may be applied in the same manner as described above.

[0040] As we age, the skin's role in separating the individual from the outside world and its regenerative capacity decline. Skin function is maintained by epidermal stem cells located in the basal layer of the epidermis and their normal differentiation. The progeny cells of epidermal stem cells adhere perpendicularly to the skin surface and arrange themselves to form a layer of a certain thickness or more, undergoing normal keratinization. This maintains the skin's regenerative capacity while also providing a strong barrier against the outside world, making it resistant to erosion and ulceration. However, with age or various stresses, these functions begin to fail, exhibiting common characteristics, and these changes are collectively referred to as skin aging.

[0041] As we age, the skin loses its fine texture, becomes uneven, and develops fine lines and wrinkles. The barrier function of the epidermis weakens, making it prone to dryness, and the epidermis, dermis, and / or adipose tissue thin or weaken, making it more susceptible to redness, eczema, sores, erosions, and ulcers. In addition, variations in skin pigmentation become more noticeable. Generally, wrinkle formation due to a decrease in collagen in the dermis, thinning (atrophy) of the epidermis, dermis, and adipose tissue, decreased firmness and weakening are observed, as well as age spots, senile lentigines, or depigmented spots, which are well known characteristics of aging skin. The inventors have found that common features in aging skin of mice and humans include histologically observed epidermal atrophy, a decrease in pigment cells, a decrease or immaturity of hemidesmosome components in the basement membrane, particularly a decrease in COL17A1 and hemidesmosomes, as well as the disappearance of fibroblasts in the upper dermis, or wrinkles and fine lines due to skin dryness, and PDGFRα in the upper dermis. + A decrease in mesenchymal cells was also observed.

[0042] It is known that deficiency of COL17A1 makes the skin fragile, and the above findings can explain why wrinkles, erosions, and ulcers are more likely to occur in aged skin. Therefore, it is understood by those skilled in the art that skin aging can be suppressed by using substances that induce or maintain the expression of COL17A1 in cells, substances that suppress the degradation of COL17A1 in cells, substances that suppress genomic stress 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 the following skin aging. (a) Thinning, weakening, atrophy, or loss of firmness of the epidermis, dermis, and / or adipose tissue (b) Impaired 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 fine wrinkles or crepe wrinkles due to dry skin. (e) Wrinkle formation due to a decrease in collagen in the dermis (f) age spots, lentigines, or depigmented spots One embodiment of the present invention is a composition for use in suppressing wrinkles.

[0044] One embodiment of the present invention relates to a composition for maintaining the regenerative capacity or competitive self-renewal capacity of epidermal stem cells, characterized in that it contains as an active ingredient 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 stress or oxidative stress in cells, and / or a substance that inhibits 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, decreased barrier function, dryness, weakening of the basement membrane, wrinkle formation due to a decrease in collagen in the dermis, thinning, atrophy, loss of firmness, weakening of the epidermis, dermis and / or adipose tissue, age spots or senile lentigines. Epidermal stem cells are stem cells that reside in the basal layer of the epidermis and supply new epidermal keratinocytes, playing an important role in skin metabolism and maintaining the skin surface in a healthy state. As epidermal stem cells age due to factors such as aging, various genomic stresses, oxidative stress, and rapid blockage of self-renewal signals by molecular targets, their inherent self-renewal capacity decreases, making them more susceptible to being lost through terminal differentiation.

[0045] As described above, the inventors have revealed that skin homeostasis is maintained by epidermal cell competition dynamics linked to COL17A1-mediated proliferation of epidermal stem cells, thereby controlling the aging of skin organs. Epidermal stem cells with high COL17A1 expression can be said to have high regenerative capacity. Therefore, those skilled in the art will understand that the regenerative capacity of epidermal stem cells can be enhanced by using substances that induce or maintain COL17A1 expression in cells, substances that suppress the degradation of COL17A1 in cells, substances that suppress genomic stress or oxidative stress in cells, and / or substances that suppress DNA damage in cells. By enhancing the regenerative capacity of epidermal stem cells and maintaining or improving the supply of new epidermal keratinocytes, skin aging is suppressed 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 anti-wrinkle use, characterized by 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 inhibits the degradation of COL17A1 in cells, a substance that inhibits genomic stress or oxidative stress in cells, and a substance that inhibits DNA damage in cells. Anti-wrinkle effects include 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] Improvement of skin elasticity In this 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 period of time). Skin elasticity is considered one indicator of skin age, and moist, springy skin feels both "soft" and "firm." Ultraviolet rays are thought to affect the decrease in skin elasticity, and the decrease in elasticity is particularly noticeable in sun-exposed areas such as the face. Skin elasticity is also called viscoelasticity, and it can be quantitatively measured using the Cutometer MPA580 (manufactured by Courage+Khazaka, Germany, by Integral Co., Ltd.) by measuring the process from when force is applied to deform the skin until it returns to its original position. The Cutometer is a commonly used instrument in the fields of dermatology and cosmetic science, and its principle is to apply negative pressure using a probe with a 2 mm diameter hole to suction the skin, then release the negative pressure and measure the degree to which the skin returns to its original shape to measure viscoelasticity. The following values ​​were used in the analysis; generally, a higher value indicates better elasticity, and these values ​​decrease with age.

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

[0049] Improvement of stratum corneum function Improving stratum corneum function refers to enhancing moisturizing ability by increasing or retaining the amount of water in the stratum corneum when applied to the skin, and improving barrier function by suppressing the intrusion of foreign substances from the outside world. A stratum corneum function improving agent is one that can exert any of these effects.

[0050] Improving skin moisturizing ability In this invention, "moisturizing ability" refers to the retention of stratum corneum moisture content, and the higher the stratum corneum moisture content, the better the moisturizing ability. A skin moisturizing ability improving agent is one that can retain this stratum corneum moisture content.

[0051] Improvement of skin barrier function Furthermore, "barrier function" refers to the suppression of transepidermal water loss, and the lower the transepidermal water loss (TEWL), the better the barrier function. The skin barrier function prevents moisture evaporation from the body and the entry of foreign substances from outside the body, and this function is impaired by ultraviolet (UV) radiation. Generally, stratum corneum water content, TEWL (the skin's water-to-wall thickness), and epidermal thickness are considered indicators of skin barrier function and show the degree of skin damage caused by UV radiation. Skin barrier function improving agents are those that can exert either the effect of preventing moisture evaporation or the effect of preventing the entry of foreign substances from outside the body.

[0052] Improving rough skin Skin roughness generally refers to a condition where the smoothness of the skin surface is lost, resulting in dryness and other problems, in contrast to healthy skin that is otherwise smooth and moisturized. These problems can include superficial issues such as a rough texture, redness, and unevenness, and may also be accompanied by itching. Skin roughness is known to be closely related to a decrease in skin barrier function. Therefore, a method of quantitatively analyzing and evaluating skin roughness symptoms is being employed, using TEWL (Thorough Skin Wetness), an indicator of skin barrier function. Furthermore, since roughness (irregularity) of the stratum corneum surface occurs with skin roughness, skin roughness can be quantitatively analyzed using numerical values ​​of ISO standard surface roughness parameters such as arithmetic mean roughness (Ra), maximum height (Ry), and ten-point mean roughness (Rz). Skin surface imaging is performed using the PRIMOS-CR three-dimensional high-resolution imaging system, and surface analysis can be performed using dedicated software (Primos OMC3_22). The inventors used a skin roughness model that serves as a standard for chemical-induced skin damage in humans and found that a substance that maintains the self-renewal ability of epidermal stem cells improves skin roughness by suppressing the increase in TEWL associated with artificially induced skin roughness and improving the roughness of the skin surface.

[0053] Accordingly, 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 suppresses genomic stress or oxidative stress in cells, and a substance that suppresses DNA damage in cells. Skin roughness refers to a condition in which the skin loses its smoothness, moisture, and even appearance and texture, often accompanied by dryness, itching, redness, and erosion. The composition of the present invention can improve the above symptoms or conditions.

[0054] Anti-spot composition A blemish refers to the deposition of melanin, a pigment produced in the skin. While "solar lentigines" (age spots) caused by prolonged exposure to ultraviolet light are the most common, the term broadly refers to any condition where the uniformity of pigmentation is disrupted, including freckles, post-inflammatory hyperpigmentation, and melasma. Therefore, "anti-blemish" means maintaining the uniformity of pigmentation. The present invention provides a composition for use in anti-blemish 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 suppresses genomic stress or oxidative stress in cells, and a substance that suppresses DNA damage in cells.

[0055] Agents for preventing and improving skin damage caused by anticancer drugs. One embodiment of the present invention relates to a method and composition for preventing or improving skin disorders associated with anticancer drug treatment.

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

[0057] In addition to skin disorders commonly observed with anticancer drugs (such as dry skin, atrophy, hand-foot syndrome, pigmentation abnormalities, and hair loss), skin toxicity from molecularly targeted drugs (various multi-tyrosine kinase inhibitors, EGF receptor inhibitors, etc.) frequently causes erythematous papules, acneiform rashes, seborrheic dermatitis, increased pigmentation of hair and nails, paronychia, dry and cracked skin, xerosis, itching, and severe stinging. Skin disorders are also observed with newer immunotherapies (immune checkpoint inhibitors). These skin disorders caused by molecularly targeted drugs are not simply considered harmful; they are evaluated as manifestations of pharmacological effects on skin cells expressing molecules common to cancer cells, or as indicators of the effectiveness of anticancer drugs. Anticancer treatment continues while symptomatic treatment with moisturizers is continued. However, these disorders cause significant psychological distress and burden for patients, and in many cases, patients abandon treatment on their own. Furthermore, severe skin disorders often lead to the discontinuation of anticancer drug administration (chemotherapy and molecularly targeted drugs), but there are no appropriate preventive or local treatment methods.

[0058] To date, it has been unclear how COL17A1 expression in epidermal stem cells is altered by many different anticancer drugs, how molecularly targeted drugs reduce or suppress this expression, and the relationship with anticancer drugs. Although symptomatic treatments such as applying moisturizers and steroids to the skin have been attempted, no effective method has been developed to prevent or improve skin damage associated with cancer treatment.

[0059] In this invention, we have revealed that not only anticancer drugs such as hydroxyureas, but also molecularly targeted drugs such as EGF receptor inhibitors, can cause a decrease in COL17A1 expression and reduce the competitive self-renewal capacity of stem cells. Therefore, the present invention provides a composition for use in preventing or improving skin disorders 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 suppresses genomic stress or oxidative stress in cells, and a substance that suppresses DNA damage in cells.

[0060] In one embodiment, the preventive and ameliorative agent for skin disorders according to the present invention may be characterized by containing as an active ingredient a substance that maintains the expression and self-renewal ability of COL17A1 in cells. More specifically, the preventive and ameliorative agent for skin disorders according to the present invention may include 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 skin disorder prevention and improvement agent according to the present invention may include apocynin, ebselen, celecoxib, apigenin, Y-27632, lipasudil, 1400W, astaxanthin, ethinylestradiol, biochanin A, necrostatin 1, Rhodiola rosea extract, Mulberry extract, Gymnema sylvestris extract, Tea extract, Loquat leaf extract, Milk thistle extract, Black turmeric extract, Plantago major seed extract, Coffee seed extract, Prunus yedoensis leaf extract, Melia azadirachta leaf extract, Mandarin orange peel extract, Lavandula angustifolia flower extract, Sophora flavescens root extract, Rice bran extract, Rosa canina fruit extract, Haemaphyllosa leaf extract, Eucalyptus globulus leaf extract, Crataegus monogyna extract, and Carrot extract.

[0061] plant extract In the present invention, various plant extracts can be used for the aforementioned applications (listed below). • To promote the healing of skin wounds, or to prevent or improve skin ulcers. • Suppression of skin aging • Enhance the regenerative capacity of epidermal stem cells • Prevention or improvement of skin disorders caused by anticancer drugs • Anti-wrinkle (improvement of skin elasticity, improvement of stratum corneum function, improvement of skin moisturizing ability, and improvement of skin barrier function) • Anti-blemish Furthermore, in this invention, plant extracts can also be used as COL17A1 promoters and hair loss / graying inhibitors.

[0062] Examples of plant extracts used in this invention include Apocynum venetum, Morus alba, Gymnema sylvestre, Camellia sinensis, Silybum marianum, Eriobotrya japonica, Kaempferia parviflora, Panax ginseng, and Indian ginseng. The parts of these plants used are not limited to those that can be used to obtain the above-mentioned effects.

[0063] The extraction method for each plant extract is not particularly limited and can be carried out according to methods well known to those skilled in the art. Water, alcoholic solvents, and organic solvents such as acetone, esters, polyhydric alcohols, and ethers can be used as extraction solvents. Therefore, for example, ethanol extracts, hot water extracts, and 1,3-butylene glycol extracts of the above plant raw materials can be prepared. These solvents may be used alone or in combination. The amount of 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 extracted liquid, a concentrate obtained by concentrating the filtrate, a dried product obtained by drying the concentrate, or a crude or purified product thereof. The concentration and drying methods can be any method. Excipients such as dextrin may be added if necessary. Purification can be carried out using synthetic adsorption resins, activated carbon, ion exchange resins, column chromatography, recrystallization, or other methods known to those skilled in the art. Furthermore, this composition can take the form of a food composition, a pharmaceutical composition, or a cosmetic composition, and in particular, in the case of a food composition, it can take the form of a functional food or a health food.

[0065] Pharmaceutical composition 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 the healing of skin wounds or in preventing or improving skin ulcers or pressure ulcers, characterized in that it contains as an active ingredient 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 suppresses genomic stress or oxidative stress in cells, and / or a substance that suppresses DNA damage in cells.

[0066] The pharmaceutical compositions according to the present invention can be used for the treatment of acute skin wounds or for the prevention or treatment of chronic skin wounds. In other words, one embodiment of the present invention relates to the use 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 suppress genomic stress or oxidative stress in cells, and / or substances that suppress DNA damage in cells, in the manufacture of pharmaceuticals for the promotion of skin wound healing or for the prevention or improvement of skin ulcers or pressure ulcers.

[0067] Furthermore, one embodiment of the present invention relates to a pharmaceutical composition for use in suppressing skin aging, characterized in that it 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.

[0068] The pharmaceutical compositions according to the present invention can be used to prevent or treat 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 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 suppress genomic stress or oxidative stress in cells, and / or substances that suppress DNA damage in cells, in the manufacture of a pharmaceutical for use in suppressing skin aging. Furthermore, one embodiment of the present invention relates to a pharmaceutical composition for enhancing the regenerative capacity of epidermal stem cells, characterized in that it contains as an active ingredient 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 suppresses genomic stress or oxidative stress in cells, and / or a substance that suppresses DNA damage in cells.

[0069] The pharmaceutical composition according to the present invention can be used to prevent or treat skin symptoms of diseases accompanied by a decrease in the regenerative capacity of epidermal stem cells. In other words, one embodiment of the present invention relates to the use 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 capacity of epidermal stem cells, substances that suppress genomic stress or oxidative stress in cells, and / or substances that suppress DNA damage in cells, in the manufacture of pharmaceuticals or regenerative medicine products (processed cells, processed cell sheets, etc.) for enhancing the regenerative capacity (self-renewal capacity) of epidermal stem cells.

[0070] Furthermore, one embodiment of the present invention relates to a pharmaceutical composition that is resistant to molecularly targeted drugs that impair the regenerative capacity (self-renewal and maintenance) of epidermal stem cells, and is characterized by containing 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 capacity 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.

[0071] Furthermore, one embodiment of the present invention relates to a pharmaceutical composition for use in preventing or improving skin disorders caused by anticancer drugs, characterized in that it contains as an active ingredient 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 suppresses genomic stress or oxidative stress in cells, and / or a substance that suppresses DNA damage in cells.

[0072] The pharmaceutical compositions according to the present invention can be used to prevent or improve skin disorders caused by anticancer drugs. In other words, one embodiment of the present invention relates to the use 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 suppress genomic stress or oxidative stress in cells, and / or substances that suppress DNA damage in cells, in the manufacture of a pharmaceutical for use in preventing or improving skin disorders caused by anticancer drugs.

[0073] Furthermore, one embodiment of the present invention relates to a pharmaceutical composition for use in anti-wrinkle purposes, characterized in that it contains as an active ingredient 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 suppresses genomic stress or oxidative stress in cells, and / or a substance that suppresses DNA damage in cells.

[0074] The pharmaceutical compositions according to the present invention can be used for anti-wrinkle purposes. In other words, one embodiment of the present invention relates to the use of substances in the manufacture of pharmaceuticals for anti-wrinkle purposes 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 suppress genomic stress or oxidative stress in cells, and / or substances that suppress DNA damage in cells.

[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 an active ingredient 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 suppresses genomic stress or oxidative stress in cells, and / or a substance that suppresses DNA damage in cells.

[0076] The pharmaceutical composition according to 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 inhibits 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 pharmaceutical for use in improving rough skin.

[0077] The pharmaceutical compositions according to the present invention can be used for anti-blemish purposes. In other words, one embodiment of the present invention relates to the use 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 suppress genomic stress or oxidative stress in cells, and / or substances that suppress DNA damage in cells, in the manufacture of pharmaceuticals for anti-blemish purposes.

[0078] In the pharmaceutical composition according to the present invention, the substance acting as the active ingredient may be, for example, a ROCK inhibitor, NADPH oxidase inhibitor, iNOS inhibitor, COX inhibitor, and ELANE inhibitor, as described in other parts of this specification, or more specifically, Y27632, apocynin, and siberestat, but is not limited thereto.

[0079] The pharmaceutical composition according to the present invention can take any form, such as tablets, powders, liquids, or semi-solids. The pharmaceutical composition according to the present invention may also be a topical drug, such as a ointment used for local treatment applied through the skin or directly to skin lesions, and can be prepared by compounding various main ingredients with a base. In addition to the above-mentioned active ingredients, the pharmaceutical composition according to the present invention may contain pharmaceutically acceptable excipients, additives, buffers, salts for isotonic adjustment, antioxidants, preservatives, drug stabilizers, etc. 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. Furthermore, the pharmaceutical composition according to the present invention may contain other active ingredients. The amount of each ingredient can be appropriately determined within a range that is pharmaceutically acceptable. Furthermore, the dosage of the composition can be appropriately determined depending on the type of drug used and the target of administration. For example, the amount of 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 target of administration. The pharmaceutical composition according to the present invention may be contained in wound dressings such as dressings. Dressings are medical materials 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 mammals, a method for preventing or improving skin ulcers or pressure ulcers, a method for suppressing skin aging, a method for enhancing the regenerative capacity of epidermal stem cells, a method for preventing or improving skin damage caused by anticancer drugs, an anti-wrinkle method, an anti-spot method, and / or a method for improving rough skin.

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

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

[0084] Pigmentation abnormality prevention and improvement agent One embodiment of the present invention relates to a method and composition for preventing or improving pigment abnormalities in aged skin. Such a method may include the steps of inducing or maintaining the expression of COL17A1 in cells, inhibiting the degradation of COL17A1 in cells, inhibiting genomic stress or oxidative stress in cells, and / or inhibiting DNA damage in cells. Such a composition may also include 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 stress or oxidative stress in cells, and / or a substance that inhibits DNA damage in cells.

[0085] One embodiment of the present invention relates to a method and composition for preventing or improving wrinkles, fine lines, dryness, and sores caused by age-related changes in the skin. Such a method may include steps of inducing or maintaining the expression of COL17A1 in cells, inhibiting the degradation of COL17A1 in cells, inhibiting genomic stress or oxidative stress in cells, and / or inhibiting DNA damage in cells. Such a composition may include 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 stress or oxidative stress in cells, and / or a substance that inhibits DNA damage in cells.

[0086] Stem cell competitive regulators One embodiment of the present invention relates to a method and composition for use in controlling cell competition in epidermal stem cells. The inventors have found that COL17A1 is an important factor involved in cell competition. Therefore, the method according to the present invention may include the steps of inducing or maintaining the expression of COL17A1 in cells, suppressing the degradation of COL17A1 in cells, suppressing genomic stress or oxidative stress in cells, and / or suppressing DNA damage in cells. Furthermore, the composition according to the present invention may include 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. 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 that can maintain 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 include 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 according to the present invention may contain apocynin, ebselen, celecoxib, apigenin, Y-27632, lipasudil, 1400W, ethinylestradiol, biochanin A, necrostatin 1, Rhodiola rosea extract, Mulberry extract, Gymnema sylvestris extract, Tea extract, Loquat leaf extract, Milk thistle extract, Black turmeric extract, Plantago major seed extract, Coffee seed extract, Prunus yedoensis leaf extract, Melia azadirachta leaf extract, Mandarin orange peel extract, Lavender flower extract, Sophora flavescens root extract, Rice bran extract, Rosa canina fruit extract, Hamemaris radiata leaf extract, Eucalyptus globulus leaf extract, Hawthorn extract, and Carrot extract.

[0088] The beauty supplement or functional food according to the present invention may take any form, such as tablets, powders, semi-solids, jellies, or liquids. The beauty supplement according to the present invention may further contain at least one of the following: skin anti-aging agents, vitamins, collagen, and minerals. The beauty supplement or functional food according to the present invention may be prepared, for example, to be taken orally 1 to 3 times a day, before, during, or after meals.

[0089] Cosmetic composition One embodiment of the present invention relates to a cosmetic composition characterized by containing as an active ingredient 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. 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, as described elsewhere in this specification. Substances that induce or maintain COL17A1 expression in cells include, for example, apocynin, ebselen, celecoxib, apigenin, Y-27632, lipasudil, 1400W, astaxanthin, ethinylestradiol, biochanin A, necrostatin 1, Rhodiola rosea extract, Mulberry extract, Gymnema sylvestris extract, Tea extract, Loquat leaf extract, Milk thistle extract, Black turmeric extract, Plantago major seed extract, Coffee seed extract, Prunus yedoensis leaf extract, Melia azadirachta leaf extract, Mandarin orange peel extract, Lavandula angustifolia flower extract, Sophora flavescens root extract, Rice bran extract, Rosa canina fruit extract, Haemaphyllosa leaf extract, Eucalyptus globulus leaf extract, and Crataegus monogyna extract.

[0090] In one embodiment, the cosmetic composition according to the Disclosure is a composition for use in skincare. In one embodiment, the cosmetic composition according to the Disclosure relates to a composition for application to the skin surface. The composition may take various product forms, but is not limited to, solutions, suspensions, lotions, creams, gels, toners, sticks, pencils, sprays, aerosols, ointments, cleansing detergents and cleansing sticks, shampoos and hair conditioners, pastas, foams, powders, mousses, shaving creams, wipes, strips, patches, electric patches, wound dressings and adhesive bandages, hydrogels, film-forming products, face and skin masks (with and without insoluble sheets), foundations, eyeliners and eyeshadows, and other makeup products.

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

[0092] The cosmetic compositions relating to this disclosure may further contain at least one of the following: a skin anti-aging agent, a skin tone enhancer, an anti-inflammatory agent, and a sunscreen. Examples of skin anti-aging agents include, but are not limited to, peptides having anti-aging properties (WO2014157485A1). Suitable skin tone enhancers include, but are not limited to, sugar amines, arbutin, deoxyarbutin, hexylresorcinol, kojic acid, hexamidine compounds, salicylic acid, and retinoids including retinyl propionate and retinyl propionate.

[0093] Suitable anti-inflammatory agents include, but are not limited to, non-steroidal anti-inflammatory drugs (NSAIDs such as ibuprofen and naproxen), salts such as glycyrrhizic acid (also known as glycyrrhetinic acid glycoside) and dipotassium glycyrrhizate, glycyrrhetenic acid, licorice extract, bisabolol (e.g., alpha-bisabolol), manjista (extracted from plants of the genus Rubia, especially Rubia cordifolia), guggal (extracted from plants of the genus Commiphora, especially Commiphora mukul), cola extract, chamomile, red clover extract, and whip coral extract (extracts from plants of the order Capricornus), 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-diphenyl acrylate, 2-ethylhexyl salicylate, and glyceryl-p-aminobenzoate. Examples include, but are not limited to, benzoates, 3,3,5-tri-methylcyclohexyl salicylate, methyl anthranilate, p-dimethylaminobenzoic acid or aminobenzoates, 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 relating to this disclosure contains at least one additive that has been conventionally used in the field of cosmetics and does not affect the properties of the composition relating to this disclosure, such as a thickener, fragrance, pearlescent agent, preservative, sunscreen, anionic or nonionic or cationic or amphoteric polymer, protein, protein hydrolysate, such as 18-methyleicosanoic acid, vitamin, panthenol, silicone, fatty acids such as vegetable oil, animal oil, mineral oil or synthetic oil, gelling agent, antioxidant, solvent, filter, etc. The composition may also contain cleaning agents, odor absorbers, colorants, abrasives, absorbents, fragrances, and other cosmetic ingredients, dyes, pigments / colorants, essential oils, anticaking agents, defoamers, antimicrobial agents, binders, biological additives, buffers, fillers, chelating agents, chemical additives, cosmetic astringents, cosmetic biocides, denaturants, drug astringents, emollients, topical analgesics, film-forming agents or materials, opacifiers, pH adjusters, preservatives, sprays, reducing agents, scavenging agents, skin coolants, skin protectants, thickeners, viscosity modifiers, vitamins, and combinations thereof. These additives may be present in the composition according to the disclosure in amounts that are not limited, but preferably or advantageously within the range of 0 to 50% by weight, 5 to 40% by weight, or 30 to 50% by weight, with respect to the total weight of the composition.

[0096] The cosmetic compositions relating to this disclosure may, for topical application to the skin, be in particular aqueous or oily solutions, lotion or serum-type dispersions, emulsions having a milky liquid or semi-liquid consistency obtained by dispersion of a fatty phase in an aqueous phase (O / W) or vice versa (W / O), suspensions or emulsions having an aqueous or anhydrous gel or cream-type consistency, or other forms such as microcapsules or fine particles, ionic and / or nonionic vesicle dispersions, or foamy forms. These compositions are prepared according to conventional methods. The compositions preferably have an aqueous phase between 5 and 99.5%.

[0097] The pH of the cosmetic composition relating to this 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, for example, ammonia, sodium hydroxide, potassium hydroxide, or a base (organic or inorganic) of a primary, secondary or tertiary (poly)amine such as monoethanolamine, diethanolamine, triethanolamine, isopropanolamine or 1,3-propanediamine to the composition, or by adding 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 citric acid.

[0098] The cosmetic compositions relating to this disclosure include, in particular, creams (e.g., day creams, night creams, anti-aging creams, moisturizing creams, makeup remover creams, foundation creams, or sun creams) for washing, protecting, treating, or caring for the face, hands, feet, folds of major body structures, or body; liquid foundations; makeup remover milks; protective or caring body milks; sun milks; lotions; gels or foams for skin care, e.g., cleansing lotions, sun lotions, artificial tanning lotions; bath compositions; deodorizing compositions containing antibacterial agents; aftershave gels or lotions; hair removal creams; and compositions for treating insect bites.

[0099] The cosmetic compositions relating to this disclosure may be applied at least once a day, twice a day, or more frequently during the treatment period. When applied twice a day, there should be an interval of at least 1 to 12 hours between the first and second applications. Typically, the compositions may be applied in the morning and / or at night before bedtime.

[0100] Screening method This invention relates to a method for screening substances effective in promoting skin wound healing or preventing or improving skin ulcers, substances effective in inhibiting skin aging, controlling cell competition, improving (enhancing, strengthening) the regenerative capacity of epidermal stem cells, preventing or improving skin damage caused by anticancer drugs, anti-wrinkle, anti-spot, and / or improvement of rough skin. Here, the inhibition of skin aging may also refer to the inhibition of aging of hair follicles, which are appendages of the skin. Such a screening method may include the following steps i-iii: i) A step of bringing cells and the test substance into contact in vitro. ii) A step of measuring the expression of COL17A1 in cells, and iii) A step of determining whether the test substance increases the expression of COL17A1. Here, the expression of COL17A1 can be measured using any method known to those skilled in the art. The substances identified by the above screening method are useful as components for promoting skin wound healing, components for suppressing skin aging, components for controlling cell competition, components for enhancing the regenerative capacity of epidermal stem cells, components for preventing or improving skin damage caused by anticancer drugs, anti-wrinkle components, anti-spot components, and / or components for improving rough skin, according to the present invention. Furthermore, they are also useful as substances to be administered to mammals in the methods for promoting skin wound healing, suppressing skin aging, enhancing the regenerative capacity of epidermal stem cells, preventing or improving skin damage caused by anticancer drugs, anti-wrinkle methods, anti-spot methods, and / or improving rough skin, according to the present invention.

[0101] The above screening method may further include a step of determining whether the test substance promotes colony formation. By combining the evaluation of COL17A1 expression with a colony formation assay, agents that promote "stem cell amplification via stem cell competition" can be screened by evaluating agents that maintain self-renewal capacity by maintaining the continuous expression of COL17A1-. That is, in one embodiment, the screening method may include the following steps i to iv: i) A step of bringing cells and the test substance into contact in vitro. ii) A step to measure the expression of COL17A1 in cells, iii) A step of determining whether the test substance increases the expression of COL17A1, and iv) A step of determining whether the test substance promotes colony formation.

[0102] Methods and kits 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 indicator. The inventors have found that the expression of COL17A1 is decreased 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 measurement of mRNA of the gene encoding COL17A1 by RT-PCR. Skin aging can be evaluated by comparing the measurement results with a control or by comparing them over time. For example, if the expression level of COL17A1 is decreased as a result of the comparison, it can be determined that skin aging is progressing.

[0103] Furthermore, 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 indicator. The kit includes, for example, an antibody that specifically recognizes COL17A1. Such an antibody may be directly labeled or recognized by a labeled secondary antibody. Labeling may be based, for example, on enzymatic color development or chemiluminescence. The kit may also include, for example, oligonucleotides (probes, primers) for detecting the transcript of COL17A1. Such oligonucleotides can be used for detection by hybridization with the transcript or in amplification reactions such as PCR. Thus, one embodiment of the present invention relates to a kit for use in a method for evaluating skin aging, comprising an antibody for detecting COL17A1 (anti-COL17A1 antibody), and a probe or primer for the gene encoding COL17A1. Furthermore, the kit may also include components (antibodies, probes, primers, etc.) for measuring the expression level of other marker proteins such as p16Ink4a.

[0104] Methods and kits 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 indicator. The inventors have found that there is a correlation between the proliferative capacity of epidermal stem cells and the expression level of COL17A1. Therefore, COL17A1 can be used as an evaluation marker for epidermal stem cells, and epidermal stem cells can be evaluated by measuring the expression of COL17A1 in epidermal stem cells. Epidermal stem cells that express COL17A1 at high levels have high proliferative capacity and can therefore 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, or FACS. Furthermore, the inventors have found that the expression of COL17A1 is decreased 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. Furthermore, epidermal stem cell aging refers to the phenomenon in which the inherent self-renewal ability of epidermal stem cells decreases due to aging, various genomic stresses, and oxidative stresses, making them more susceptible to loss during terminal differentiation. For example, if the expression level of COL17A1 is considered high when compared to a standard value, the cells are judged to be suitable for use in culture and regenerative medicine products.

[0105] Furthermore, 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 indicator. This kit may include, for example, an antibody that specifically recognizes COL17A1, a probe for the gene encoding COL17A1, or a primer for amplifying the 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, probe, or 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 indicator. The inventors have found a correlation between the proliferative capacity of epidermal stem cells and the expression level of COL17A1. Epidermal stem cells with high COL17A1 expression levels have high proliferative capacity and are therefore suitable for use in culture. Thus, COL17A1 can be used as a marker for evaluating the proliferative capacity of epidermal stem cells, and measuring the expression of COL17A1 in epidermal stem cells and selecting epidermal stem cells with high COL17A1 expression levels can be beneficial when subsequently performing culture or transplantation. The selection of cells that highly express COL17A1 can be performed by any method known to those skilled in the art, for example, by 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 indicator. This kit may, for example, include 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] Methods and evaluation kits for amplifying epidermal stem cells and / or epidermal cells. One embodiment of the present invention also relates to a method for amplifying epidermal stem cells and / or epidermal cells, which includes a step of selecting epidermal stem cells with high COL17A1 expression. Epidermal stem cells with high COL17A1 expression have high proliferative capacity, and by selecting such cells, epidermal stem cells and / or epidermal cells can be efficiently amplified. High COL17A1 expression in epidermal stem cells means that the expression level is quantitatively higher compared to that in aging cells. For example, they can be isolated using methods such as FACS or bead-based methods. Cell sheets can be prepared ex vivo using such amplification methods.

[0109] Furthermore, one embodiment of the present invention relates to a kit for use in a method for amplifying epidermal stem cells and / or epidermal cells, comprising a step of selecting epidermal stem cells with high expression levels of COL17A1. The kit may, for example, include 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 amplifying epidermal stem cells and / or epidermal cells, comprising an antibody, probe, or primer for detecting COL17A1.

[0110] Methods and kits 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 reduce such competition, and substances that alter the efficiency or rate of competition, by altering the expression levels of molecules that modify the expression of molecules involved in cell competition, such as COL17A1, in some cells. The inventors have found that the expression level of COL17A1 correlates with cell competition in epidermal stem cells. Epidermal stem cells with high COL17A1 expression levels have an advantage in cell competition. Furthermore, the evaluation of cell competition can be performed using three-dimensional culture of epithelial cells that form stratified squamous epithelium, such as keratinocytes (including cultured keratinocytes and keratinocyte cell lines such as HaCat cells and Pam212 cells). Cells with high COL17A1 expression levels proliferate more favorably than cells with low COL17A1 expression levels, and can therefore be evaluated as having high cell competitive ability.

[0111] Furthermore, the present invention allows for the evaluation of cell competition in three-dimensional culture of keratinocytes and the search for compounds, extracts, nucleic acids, etc., that control this competition. Therefore, one embodiment of the present invention relates to a method for evaluating cell competition in keratinocytes, and more particularly to a screening method for substances that control cell competition, using cell competition in three-dimensional culture of keratinocytes as an indicator.

[0112] One embodiment of the present invention relates to a kit for use in an evaluation method of cell competition in keratinocytes (keratinocytes). This kit includes, for example, nucleic acids that change the expression of cell competition-related molecules such as COL17A1 and cancer-related genes, such as nucleic acids for use in RNA interference (siRNA, shRNA, etc. against the gene encoding COL17A1), as well as gene transfection reagents, lentiviruses or retroviruses, antibodies for immunostaining such as antibodies that specifically recognize COL17A1, and devices for three-dimensional culture. Therefore, one embodiment of the present invention relates to a kit for use in an evaluation method of cell competition, including cell lines, viruses, antibodies, and devices related to the evaluation of cell competition in keratinocytes. Further, this kit may 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. Isolation of cells expressing COL17A1 can be performed by any method known to those skilled in the art, for example, by methods using FACS or beads. Further, the cell composition of the present invention may include those having a three-dimensional structure such as an epithelial sheet or an organoid.

[0114] It is desirable that the cells in this cell composition express COL17A1 at a high level. Therefore, the cell composition of the present invention includes those treated with a substance that induces or maintains the expression of COL17A1 in cells. Cells expressing COL17A1 at a high level (COL17A1 high cells) can be isolated, for example, using FACS. Here, the terms "high" or "hi" representing the expression level are well known in the art, and "COL17A1 high"High" indicates a high level of COL17A1 expression compared to the cell population being analyzed. In the context of this disclosure, "high" or "hi" may be considered to represent an expression level within the top 50% of the target cell population, for example, 3%, 5%, 10%, 15%, 20%, 30%, or 40%. The cells in this cell composition may be, for example, epidermal keratinocytes or mucosal epithelial keratinocytes, but other cells may also be included. For example, at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the cells in this cell composition may express COL17A1. This cell composition contains at least 1 × 10⁶ cells. 3 pieces, 1×10 4 pieces, 1×10 5 pieces, 1×10 6 pieces, 1×10 7 pieces, or 1 × 10 8 It may contain individual cells.

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

[0116] The cells in this 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 containing cells expressing COL17A1. Such a cell sheet can be prepared by isolating and culturing cells expressing COL17A1. Such a cell sheet can be transplanted into a patient for use in treating the patient's disease or injury. The cell sheet may 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 in any way by these examples. [Examples]

[0119] Example 1: Age-related decrease in COL17A1 protein expression in the skin Unlike the skin on the back, the tail skin of mice is characterized by a layered epidermal cell layer, and as observed in the histological analysis of human epidermis, the tail skin of aged mice is also characterized by atrophy with a decrease in the number of spinous cells in the scaly epidermis. The basal cells of young mice have a rectangular, elongated shape relative to the basement membrane, while aged basal cells have a more rounded or flatter shape. In aged skin, the total number of basal cells and MCM2 + A significant decrease in non-resting basal cells was observed, indicating that epidermal stem cells and their cell division decline with age. Ultrastructural analysis of epidermal basal cells and the epidermal-dermal junction using transmission electron microscopy (TEM) revealed a significant decrease in the number of electron-density regions (hemidesmosomes; HDs) that anchor the basal cell layer to the basement plate with age. Furthermore, cell detachment from basal cells was frequently observed in the lamina lucida (LL) of aged tail skin, which is associated with the age-related decrease in HDs from the basal cell layer.

[0120] Therefore, the inventors hypothesized that HD components are directly involved in the skin aging process. To verify this, the expression of various HD components and integrin β1 (ITGB1), a major cell adhesion molecule, was analyzed in the tail epidermis of young and older mice. As a result, it was revealed that the immunostaining level of COL17A1 significantly decreased with aging (Figure 1). Consistent with this, immunoelectron microscopy analysis also revealed a significant decrease in the signaling of COL17A1 in the dermal-epidermal junction (LL) of aged skin. Furthermore, whole-body immunostaining revealed a heterogeneous distribution of COL17A1 and ITGA6 in aged skin. In particular, COL17A1-negative cell regions expressing ITGA6 were found in addition to the double-negative cell regions of aged tail epidermis. This indicates that COL17A1 protein expression is the most unstable, and consequently, insufficient COL17A1 leads to the eventual destabilization of other HD components with age.

[0121] To investigate the role of COL17A1 in the stability of HD components, we analyzed the number of HDs in the skin of young and aged mice (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 (UV) irradiation also shows downregulation of COL17A1 expression in several basal layer keratinocytes in vitro and in vivo. As a result, UV irradiation of the skin caused HD destabilization. These data together suggest that COL17A1 functions as a stress / compatibility sensor that monitors the DNA damage response as a "stem cell checkpoint molecule," determining whether stem cells self-replicate or differentiate. This stress-sensing mechanism may lead to differences in the self-renewal capacity of epidermal stem cell clones with different levels of COL17A1 expression present in aged epidermal basal layer.

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

[0123] Next, to test the possibility that heterogeneity in basal cells with different COL17A1 expression levels induces stem cell competition in the basal layer during aging, we observed COL17A1 expression levels from multicolor-labeled stem cell clones in the interfollicular epidermis (IFE) of young (11-month-old) and aged (28-month-old) mice. As shown in Figure 4, in aged skin, COL17A1 expression in monocolor basal layer cells representing stem cell clones was generally decreased, while COL17A1 expression in basal layer cells of young mice varied in expression levels among clones and generally decreased with aging. A detailed comparison of basal layer keratinocyte clone size and COL17A1 expression levels revealed a positive correlation between basal layer cell clone size and COL17A1 expression levels in aged mouse skin (Figure 5). This indicates that stem cell clones maintaining higher levels of COL17A1 expression exhibit greater clonality and become dominant in the epidermal basal layer during the physiological aging process. Furthermore, no significant induction of apoptotic cell death or so-called “cellular senescence” was observed in basal layer cells of aged skin. These data suggest that COL17A1 high Epidermal stem cells, COL17A1 low This suggests that the cells are competing with each other to withstand epidermal aging.

[0124] Example 3: Differential expression of COL17A1 that induces competition among epidermal stem cells COL17A1 low / - The emergence of the clone in vivo is adjacent to the surrounding COL17A1 high To investigate whether direct cell competition with clones occurs, the inventors created drug-inducible Col17a1 gene knockout (cKO) mice combined with a basal layer keratinocyte-specific multicolor labeling system. Rare induction of COL17A1 deficiency using low-dose TAM administration resulted in the observation of monocolor-labeled Col17a1-deficient keratinocyte clones in the tail epidermis two days after knockout induction. In control mice, these clones increased in size by day 14 and subsequently formed a monocolor column (basal clones) by 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, 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 significant exclusion of Col17a1-deficient cell clones from the skin. These data indicate that epidermal stem cells expressing COL17A1 compete with COL17A1-deficient epidermal stem cells within the same niche, suggesting that COL17A1 mediates skin homeostasis and the aging process through a cell competition mechanism in the epidermis.

[0125] To further investigate whether differences in COL17A1 expression directly mediate cell competition in the epidermis, a novel in vitro cell competition assay system was established by expressing short hairpin (sh)RNA in 3D-cultured HaCaT human keratinocytes. EmGFP expressing shCOL17A1 + COL17A1 - Stable HaCaT cells and control EmGFP expressing shScramble. + COL17A1 +HaCaT cells were generated and cultured in a 3D culture system. Neither shScramble nor shCOL17A1-expressing HaCaT cells showed any significant differences in structure, stratification, proliferation number, or apoptotic cells in the 3D cultured epidermis. On the other hand, COL17A1 - EmGFP + Cells and wild type (COL17A1) + Co-culturing with wild-type cells in a 1:10 ratio significantly excluded shCOL17A1-expressing cells. However, no significant exclusion of COL17A1-expressing cells was detected at a 1:3 ratio. These data indicate that Col17A1-deficient basal layer cells are excluded from the basal layer as loser cells by a sufficient number of surrounding wild-type cells. In summary, these data suggest that COL17A1-mediated cell competition occurs in the physiological aging process of human epidermis, and that COL17A1 expressed by epidermal stem cells confers competitive self-renewal ability between neighboring epidermal stem cell populations.

[0126] Example 4: Maintenance of epidermal homeostasis by COL17A1-mediated SCD To understand the COL17A1-mediated cell competition mechanism, the inventors have found that COL17A1 is mediated by COL17A1 + We hypothesized that promoting SCD (Splitting Division of Degeneration) theoretically maintains the clonal proliferation of epidermal stem cells. To test this hypothesis, we analyzed the division axis of basal layer cells in vivo. Normal basal layer cells mostly exhibited cell division parallel to the basement membrane, while Col17a1-deficient basal layer keratinocytes abnormally induced vertical cell division, producing basal layer cells and progeny located above the basal layer. This indicates that COL17a1 mediates SCD, suggesting that COL17a1-mediated SCD pushes away Col17a1-deficient basal layer cells with lost or reduced HD (Hydration Degeneration), separating them from the basement membrane. Indeed, Col17a1-deficient keratinocytes eventually detach from the basal layer IFE (Infrastructure Focused Emission).

[0127] Therefore, these data are COL17A1 +We have shown that COL17A1-dependent SCD, which promotes clonal proliferation of winner cells, induces cell competition during the aging process. Importantly, basal cells in aged epidermis showed an increased rate of vertical cell division and often decreased levels of COL17A1 expression. This ultimately leads to epidermal atrophy (thinning) due to impaired cell division accompanied by loss of COL17A1 expression in aged skin areas. In stark contrast, basal cells in COL17A1 transgenic (tg) mice, in which basal cells maintained COL17A1 expression, maintained horizontal cell division relative to the basement membrane. These data suggest that COL17A1-mediated SCD generates a mechanical driving force for cell competition, allowing epidermal aging to be tolerated through competitive self-renewal phenomena as a result of horizontal cell spread and basement membrane occupancy in adult epidermis.

[0128] Furthermore, it is known that in hematopoietic stem cells, amplification of specific stem cell clones occurs predominantly, while other stem cell clones disappear, and that competitive self-renewal of stem cells occurs. The molecules involved in this are also being identified. However, the actual state of "competitive self-renewal" under physiological conditions in the epidermis is unknown, and the molecules responsible for it have not been identified.

[0129] To test whether COL17A1-mediated SCD maintains clonal proliferation of epidermal stem cell clones, we used a colony formation assay, a gold standard in vitro assay for evaluating the self-renewal capacity of epithelial stem cells, including cells in the tail epidermis. Aged epidermal keratinocytes expressing low levels of COL17A1 had reduced colony formation capacity and produced smaller colonies compared to younger tail keratinocytes (Figure 7). In contrast, forced expression of hCOL17A1 in mouse basal layer keratinocytes promoted clonal capacity of epidermal stem cells, resulting in significantly larger colony size and number compared to control mice (Figure 8).

[0130] These data indicate that COL17A1 mediates clonal proliferation through the proliferative capacity of epidermal stem cells. Consistent with this, COL17A1 expression in tg mice significantly rescued age-related epidermal thinning and age-related shedding. In summary, the COL17A1-dependent proliferative capacity of epidermal stem cells that continuously perform symmetric cell division (SCD) in colony assays suggests that COL17A1 plays a role in epidermal homeostasis and cell competition during aging. high This explains the higher compatibility of stem cell clones and suggests they possess competitive self-renewal capabilities.

[0131] Example 5: Maintenance of heterotype cell lineages by COL17A1-expressing epidermal stem cells Skin aging is characterized not only by epidermal aging but also by melanocyte-related pigmentation abnormalities and dermal changes. To study the functional significance of epidermal stem cell aging in skin aging, we evaluated whether physiological aging induces epidermal pigmentation abnormalities in the tail epidermis. Physiological aging was observed to gradually induce skin pigmentation abnormalities (heterogeneous skin pigmentation with areas of low and high pigmentation), ultimately leading to hypopigmentation in the tail skin. Detailed whole-mount microscopic observation of the tail revealed that the pigmentation pattern became heterogeneous with age. To visualize the distribution of melanocyte-derived 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 analyses showed that H2B GFP was present in the tail 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 juvenile and aged whole epidermal cells ranked melanocyte genes as the top major changes during aging. Importantly, highly pigmented melanocytes were observed in the basal layer region of 12-month-old tails, and these cells were occasionally present in the upper basal layer region, suggesting that they are detached from the basal layer and eliminated from the skin.

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

[0133] Chronic UVB irradiation induces cutaneous pigmentation abnormalities and HD damage in the epidermis of mice and humans. The inventors then investigated whether chronic UVB exposure induces cutaneous pigmentation abnormalities and HD damage, as well as their associated phenotypes in the tail epidermis (Figure 9). Following hyperpigmentation of the skin induced by repeated UVB irradiation, epidermal pigmentation abnormalities were induced within one month after the final UVB irradiation, similar to Col17a1 or ITGA6 cKO mice. Ultrastructural analysis using TEM and immunoTEM revealed that UVB irradiation inhibits the formation of mature HD and reduces COL17A1 signaling, similar to Col17a1 cKO.

[0134] These data together demonstrate that chronic UVB-mediated HD damage induces epidermal pigmentation abnormalities through damage to COL17A1-expressing epidermal keratinocytes. Furthermore, overexpression of COL17A1 in mouse basal layer keratinocytes is associated with macroscopic and microscopic pigmentation abnormalities, as well as KIT + This significantly rescued age-related decline in epidermal melanocytes (Figure 9). In summary, these data indicate that, during aging and photoaging, melanocytes within the epidermis are maintained by adjacent COL17A1-expressing epidermal stem cells within the epidermis.

[0135] The skin's regenerative capacity declines with age. The inventors investigated how age-related changes in epidermal stem cells are involved in skin wound healing, which progresses through the cloning of epidermal stem cells, and whether they affect dermal fibroblasts (Figure 10). Analysis of PDGFRα-positive mesenchymal cells showed a significant decrease in the population located just below the epidermis with aging, and that forced expression of hCOL17A1 rescued age-related loss of PDGFRα-positive cells. These data suggest that maintaining COL17A1-mediated epidermal stem cells strengthens the stability of the epidermal-dermal junction and resists aging of skin organs, including skin atrophy, fine wrinkles, and skin aging caused by its disruption.

[0136] Example 6: HD is key to skin organ regeneration. To test the above hypothesis, the inventors conducted full-thickness wound healing experiments using tail skin from aged wild-type mice. Measurements of the wound area showed that physiological aging significantly delayed the wound healing process. The inventors 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. Furthermore, forced expression of hCOL17A1 by mouse basal layer keratinocytes promoted wound healing (Figure 12). These data indicate that COL17A1-mediated clonal proliferation 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 inventors searched for novel compounds that induce COL17A1 expression in keratinocytes in vitro. As shown in Table 1 and Figure 15, the 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 an increase in the competitive self-renewal capacity of epidermal stem cells exhibiting SCD, the inventors performed a colony formation assay using human epidermal keratinocytes. Consistently, 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 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, as did tg mice overexpressing human COL17A1 (Figure 12). These results indicate that COL17A1-inducing drugs promote skin wound healing by facilitating re-epithelialization of the wound margins through the proliferation of epidermal stem cells. These findings open up new avenues for the development of novel drugs to promote skin regeneration.

[0139] Example 7: Search for substances that maintain and promote the competitive self-renewal ability of epidermal stem cells Further investigation was conducted to identify substances that induce or maintain COL17A1 expression. Human epidermal keratinocytes were seeded in 384-well plates, cultured for 48 hours with reagents added, and immunofluorescence staining was performed using anti-COL17A1 antibody and fluorescently labeled secondary antibodies. The fluorescence intensity per cell was then detected using a high-content imaging system (HCS: high-content screening). Western blotting (WB) analysis was also performed for some substances. In addition to cell counting using DAPI, the expression level of COL17A1 was quantified.

[0140] As a result, we identified a group of substances that maintain the self-renewal capacity of epidermal stem cells through COL17A1 expression. However, we were unable to adequately distinguish these substances from steroids and other cytotoxic substances that transiently increase COL17A1 expression but reduce self-renewal capacity in the long term, and these could not be excluded by measuring COL17A1 expression alone. Therefore, by combining this with a colony formation assay, we succeeded in selecting substances that promote competitive self-renewal capacity via COL17A1 over the long term. The results are shown in Figure 15 and Tables 1 and 2.

[0141] [Table 1]

[0142] In Table 1, two double circles (◎◎) for COL17A1 expression (compared to the control) indicate an increase of 2.0 times or more, one double circle (◎) indicates an increase of 1.2 times or more, and one circle (〇) indicates maintenance at 1.0-1.2 times. For the control ratio of colony number or colony area, two double circles (◎◎) indicate an increase of 1.2 times or more, one double circle (◎) indicates maintenance at 1.0-1.2 times, and X indicates 1.0 or less (the same applies below). ND (Not done) indicates that the analysis was not performed.

[0143] Many substances that induce or maintain the expression of COL17A1, which is expressed by epidermal stem cells, were found to enhance colony formation ability, i.e., the self-renewal ability of epidermal stem cells (Figure 16, Table 1, Table 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 COL17A1 expression analysis with colony formation ability, it is possible to select substances that possess "competitive self-renewal ability" in vivo.

[0144] [Table 2]

[0145] [Table 3]

[0146] Table 3 summarizes anticancer drugs and molecularly targeted drugs that show a decrease in COL17A1 expression. Conventional chemotherapy agents such as antimetabolites and alkylating agents alter COL17A1 expression. In particular, molecularly targeted drugs that target cancer cells significantly reduce COL17A1 expression (Table 3).

[0147] Example 8: Decrease in COL17A1 due to anticancer drugs and various stresses Seven-week-old C57BL6 mice were administered the anticancer drug hydroxyurea three times a month. Tissue samples were collected after three months and immunostained using an antibody against COL17A1. The results showed a significant decrease in COL17A1 expression in the basal layer of mice treated with hydroxyurea (Figure 17). In addition, human epidermal keratinocytes, HaCaT cells, were irradiated with radiation (20 Gy, 30 Gy) and ultraviolet light (20 mJ, 30 mJ). Cells were collected 24 hours and 72 hours later, respectively, and Western blotting was performed using an antibody against COL17A1. The results showed a significant decrease in COL17A1 protein levels in both radiation and ultraviolet irradiation. Furthermore, when HaCaT cells were administered hydrogen peroxide, which induces oxidative stress, and COL17A1 expression was similarly analyzed, a significant decrease was observed (Figure 17).

[0148] From the above, it became clear that COL17A1 expression decreases due to various stresses such as anticancer drugs, ultraviolet light, radiation, and oxidative stress.

[0149] Example 9: Improvement test of skin disorders caused by anticancer drugs by COL17A1 overexpression Transgenic mice overexpressing human COL17A1 (COL17A1-Tg) and control mice (approximately 18 months old) were acclimated to the experimental environment. After this, the hair on their backs was plucked under anesthesia, and they were divided into the following six groups. • Control mice (n=3) not treated with EGF receptor inhibitors • COL17A1-Tg mice, EGF receptor inhibitor non-administration group (n=3) • Control mice treated with an EGF receptor inhibitor (erlotinib) (n=4) • COL17A1-Tg mice treated with an EGF receptor inhibitor (erlotinib) (n=4) • Control mice treated with an EGF receptor inhibitor (gefitinib) (n=3) • COL17A1-Tg mice treated with an EGF receptor inhibitor (gefitinib) (n=3)

[0150] Erlotinib or gefitinib, both EGF receptor-targeted anticancer drugs, were dissolved in a 0.5% methylcellulose solution at a concentration of 5 mg / ml and administered by intraperitoneal injection at a daily dose of 50 mg / kg / day starting from the beginning of the study. The control group (non-administered group) received the same 0.5% methylcellulose solution as the solvent. Transepidermal water loss (TEWL) was measured for each group of mice on day 7 after the start of erlotinib or gefitinib administration. The measurement was performed using a Tewameter TN300 (Courage+Khazaka, Germany; Integral Co., Ltd.) at specific locations on the back of the mice (two locations symmetrically on either side of the center of the spine).

[0151] Figure 18 shows the test results. As shown in Figure 18, control mice administered with erlotinib or gefitinib showed a significant increase in TEWL, but compared to these, transgenic mice overexpressing COL17A1 showed significantly lower TEWL values ​​even when administered with erlotinib or gefitinib, suggesting that skin damage was reduced. This indicates that skin damage induced by anticancer drug administration is significantly improved by promoting the self-renewal ability of epidermal stem cells through the overexpression of COL17A1.

[0152] Example 10: Improvement test of 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, then under anesthesia, the hair on their backs was plucked, and they were divided into the following six groups (n=4) so ​​that their body weights were nearly uniform. • 7 weeks old / group not receiving EGF receptor inhibitor (erlotinib) • 7 weeks old / EGF receptor inhibitor (erlotinib) administration + control solvent application group • 7 weeks old / EGF receptor inhibitor (erlotinib) administration + application of apocynin 200 μM • 6 months old / group not receiving EGF receptor inhibitor (erlotinib) • 6 months old / EGF receptor inhibitor (erlotinib) administration + control solvent application group • 6 months old / EGF receptor inhibitor (erlotinib) administration + apocynin 200 μM application group

[0153] Erlotinib was dissolved in a 0.5% methylcellulose solution at a concentration of 5 mg / ml and administered by intraperitoneal injection at a rate of 50 mg / kg / day daily from the start of the study. The control group (non-administered) received the same 0.5% methylcellulose solution as the solvent. The control solvent application group and the apocynin 200 μM application group received aqueous solutions of each preparation once daily, five times a week, on their backs. The control solvent application group also received the apocynin solvent (50% ethanol / PBS) in the same manner. TEWL was measured for 7-week-old mice on day 7 after the start of erlotinib administration, and for 6-month-old mice on day 12, for each group of mice. Measurements were performed using a Tewameter TN300 (Courage+Khazaka, Germany; Integral Co., Ltd.) on specific sites on the back of the mice (two locations symmetrically on both sides of the spine).

[0154] Figure 19 shows the test results. As can be seen from the results in Figure 19, TEWL increased significantly in mice administered erlotinib, but in the group treated with apocynin, TEWL was significantly reduced, and skin damage was alleviated, even under erlotinib administration. This indicates that skin damage induced by molecularly targeted drug administration can be significantly improved by substances that promote the competitive activity of epidermal stem cells.

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

[0156] Example 12: Barrier function improvement test The skin barrier function is a primary function of the skin, preventing moisture evaporation from the body and the entry of foreign substances from outside. However, UV irradiation causes the epidermis to thicken reactively, while simultaneously impairing the barrier function. Generally, stratum corneum water content and TEWL are used to evaluate skin barrier function, and together with reactive epidermal thickness, they are considered indicators that reflect the degree of skin damage caused by UV radiation, anticancer drugs, etc.

[0157] After acclimatizing hairless mice HOS:HR-1 females (7 weeks old) to the experimental environment, they were divided into the following six groups to ensure nearly uniform body weight. · UV non-irradiation group (n=2) • UVB irradiation + solvent coating group (n=4) • Group treated with ultraviolet (UVB) irradiation + apocynin 200 nM (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 this case, the solution-coated group had an aqueous solution of each preparation applied to the back of hairless mice once a day for 5 days, starting the day before UV irradiation. In the "UV + solvent-coated group," the solvent (50% ethanol / PBS) of the preparation from Example 1 was applied in the same manner. UVB irradiation was performed using a Yayoi UV irradiation device (Y-UV-Lab-K-D5 lamp type) once a day at 150 mJ / cm². 2Irradiation was performed at the intensity of . On the 4th day after the start of ultraviolet irradiation, the stratum corneum water content and TEWL at the irradiated site were measured for the mice in each group. The measurement was performed on specific sites (two symmetric sites on the left and right from the center of the spine) on the back of the mice using a Corneometer CM825 and a Tewameter TN300 (manufactured by Courage+Khazaka GmbH, Integral Co., Ltd.).

[0159] Figure 21 shows the effect of substances that promote the self-renewal ability of epidermal stem cells on maintaining the stratum corneum water content against ultraviolet irradiation. The stratum corneum water content after ultraviolet irradiation showed a significantly lower value compared to the control group without ultraviolet irradiation. However, the 200 μM apocynin administration group, the 2 μM and 20 μM Y-27632 administration groups showed a significantly higher stratum corneum water content. Thus, it was revealed that substances that promote the cell competition ability of epidermal stem cells have the effect of maintaining the stratum corneum water content against ultraviolet irradiation. Furthermore, the effect of substances that promote the self-renewal ability of epidermal stem cells on suppressing TEWL against ultraviolet irradiation was analyzed. As shown in Figure 21, the TEWL after ultraviolet irradiation showed a significantly higher value compared to the control group without ultraviolet irradiation. However, the 200 μM apocynin administration group and the 20 μM Y-27632 administration group showed a significantly lower TEWL. Thus, it was revealed that substances that promote the cell competition ability of epidermal stem cells have the effect of suppressing the increase in TEWL caused by ultraviolet irradiation.

[0160] Example 13: Evaluation of improvement in skin elasticity (skin firmness) Due to changes in the state of the dermis caused by ultraviolet rays and aging, the skin loses elasticity (= firmness), and wrinkles and sagging occur. Indicators for confirming the elasticity of the skin include the elongation when the skin is stretched and the return when released, and it has been found that multiple elasticity indices decrease with aging. An increase in skin elasticity is considered to lead to an improvement effect on skin wrinkles, sagging or firmness.

[0161] After acclimating hairless mice HOS:HR-1 females (7 weeks old) to the experimental environment, they were grouped into the following 3 groups (n = 4) so that their body weight values were almost uniform. · Non-ultraviolet irradiation group · Ultraviolet (UVA + UVB) irradiation + solvent application group · Ultraviolet (UVA + UVB) irradiation + apocynin 200 μM application group

[0162] At this time, in the above solution application group, an aqueous solution of each preparation was applied to the back once a day, three times a week for 5 weeks starting from the day before UV irradiation. Also, in the "ultraviolet + solvent application group", the solvent of apocynin (50% ethanol / PBS) was applied in the same manner. For UVB irradiation, a UV irradiation device manufactured by Yayoiken Co., Ltd. (Y-UV-Lab-K-D5 lamp type) was used, and UVA was irradiated at 2 J / cm 2 and UVB was irradiated at 150 mJ / cm 2 at a strength of 5 weeks.

[0163] Skin elasticity measurements were taken on the backs of each group at the 5th week after starting UV irradiation (at two symmetric locations on the left and right with respect to the center of the spine). Skin elasticity was measured using a Cutometer MPA580 (manufactured by Courage + Khazaka GmbH, Integral Co., Ltd.) (parameter settings: 300 mba, on-time 2 seconds, off-time 2 seconds), and the average value of three measurement values was used for analysis. The skin on the mouse's back was suctioned for 2 seconds, and then the suction was released 2 seconds later. The length of skin stretch at each of the time points immediately after suction, immediately before suction release, and immediately after suction release was measured, and the rapid stretchability at the initial stage of suction (Ue: unit mm), the maximum skin stretch at immediately before suction release (Uf: unit mm), and the rapid recovery amount at immediately after suction release (Ur: unit mm) were used. As an index of net elasticity, R5 = Ur / Ue was used, and as an index of elasticity, R7 = Ur / Uf was used.

[0164] As shown in Figure 22, the value of skin elasticity significantly decreases due to UV irradiation, but in the test group where a substance that promotes the self-renewal ability of epidermal stem cells was applied, the skin elasticity was significantly improved compared to the control group. The increase in skin elasticity is considered to lead to an improvement effect on skin wrinkles, sagging, or firmness. From this, it was suggested that by applying a substance that promotes the cell competition ability of epidermal stem cells, an improvement effect on skin wrinkles, sagging, or firmness can be obtained.

[0165] Example 14: Evaluation of improvement in skin elasticity (skin firmness) through 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 nearly uniform. • Control (solvent) coating group • Group coated with 200 μM apocynin • Group treated with 0.1% retinol

[0166] At this time, the groups treated with the above-mentioned solutions were given an 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 measurements and skin elasticity measurements were performed on the backs of each group (two locations symmetrically on either side of the center of the spine). TEWL measurements were performed using a Tewameter TN300 (manufactured by Courage+Khazaka GmbH, Germany, and Integral Corporation) on specific areas of the mouse backs (two locations symmetrically on either side of the center of the spine). Skin elasticity was measured using a Cutometer MPA580 (manufactured by Courage+Khazaka GmbH, Germany, and Integral Corporation) (parameter settings: 300 mba, on time 2 seconds, off time 2 seconds), and the average of three measurements was used for analysis. The skin on the back of a mouse was suctioned for 2 seconds, and then released after 2 seconds. The length of skin elongation was measured at three points: immediately after suction, immediately before suction release, and immediately after suction release. The following metrics were used: skin return upon suction release (Ua: in mm), rapid extensibility in the initial stage of suction (Ue: in mm), maximum skin extensibility immediately before suction release (Uf: in mm), and rapid recovery immediately after suction release (Ur: in mm). R5 = Ur / Ue was set as the index of net elasticity, and R7 = Ur / Uf was set as the index of elasticity. R2 = Ua / Uf was set as the index of total elasticity.

[0167] As shown in Figure 23, in the test group that received long-term, continuous application of a substance that promotes the self-renewal ability of epidermal stem cells, skin elasticity was significantly improved in all three indicators compared to retinol, which was used for comparison. Furthermore, while continuous application of retinol is thought to significantly increase the skin's TEWL and suppress barrier function, the substance that promotes the self-renewal ability of epidermal stem cells maintains normal barrier function. Therefore, continuous application of the substance that promotes the self-renewal ability of epidermal stem cells is thought to increase elasticity while maintaining normal skin barrier function, resulting in preventive and ameliorative effects against wrinkles, sagging, and loss of firmness.

[0168] Example 15: Wrinkle Improvement Test In a mouse model in which wrinkle formation was induced by long-term irradiation with ultraviolet (UVA) light, we observed the wrinkle-improving effect of a substance that promotes the self-renewal ability of epidermal stem cells.

[0169] After acclimatizing hairless mice HOS:HR-1 females (7 weeks old) to the experimental environment, they were divided into the following three groups to ensure nearly uniform body weight. · UV non-irradiation group (n=2) • Group subjected to UV (UVA + UVB) irradiation + solvent coating (n=4) • Group treated with ultraviolet (UVA + UVB) irradiation + 200 μM apocynin (n=4) • Group treated with UVA + UVB irradiation + 0.1% retinol application (n=2)

[0170] In this study, the solution application group received an aqueous solution of each preparation once daily, three times a week for five weeks, starting the day before UV irradiation, on the backs of hairless mice. The "UV + solvent application group" received a similar application of apocynin solvent (50% ethanol / PBS). Retinol, considered effective in wrinkle improvement, was applied similarly at a concentration of 0.1% to the control group. UVB irradiation was performed using a Yayoi UV irradiation device (Y-UV-Lab-K-D 5-lamp type), with UVA irradiation at 20 J / cm² three times a week. 2 , UVB 100 mJ / cm 2 The irradiation was performed at that intensity for four weeks. The backs of mice in each group were imaged using a high-resolution three-dimensional image analyzer, PRIMOS-CR (manufactured by Canfield Scientific, Inc., and operated by Integral Co., Ltd.), from week 1 to week 4 after the start of UV irradiation. Based on the obtained images, the total wrinkle volume (mm²) was calculated using Primos OMC3_22 software. 3 The mean total wrinkle depth (μm) was measured. Skin flexibility was measured using a Cutometer MPA580 (Courage+Khazaka GmbH, Germany; Integral Co., Ltd.), and the R0 value was defined as skin flexibility. The R0 value reflects the state of the stratum corneum and serves as an indicator of skin flexibility. TEWL was measured on the back of each group (two locations symmetrically on both sides of the spine) using a Tewameter TN300 (Courage+Khazaka GmbH, Germany; Integral Co., Ltd.).

[0171] As shown in Figure 24, UV irradiation significantly increases skin wrinkles, but in the test group treated with a substance that promotes the competitive ability of epidermal stem cells, wrinkle formation was suppressed compared to the control group, and the degree of suppression was more pronounced than with retinol. The total volume of wrinkles at this time (mm 3 When the average total wrinkle depth (μm) was measured, as shown in Figure 24, skin wrinkles significantly increased with UV irradiation, but they improved remarkably in the test group treated with a substance that promotes the cell competitiveness of epidermal stem cells. This suggests that applying a substance that promotes the self-renewal ability of epidermal stem cells can improve skin wrinkles.

[0172] Furthermore, when transepidermal water loss (TEWL) was measured in each group of mice, the group treated with retinol showed a significant increase in TEWL compared to the other groups (Figure 25). On the other hand, the group treated with a substance that promotes the self-renewal ability of epidermal stem cells showed no difference in TEWL values ​​compared to the control group, suggesting that wrinkles were reduced while maintaining normal barrier function.

[0173] Given the significant increase in TEWL observed with retinol, skin flexibility (R0), which is considered to reflect the condition of the stratum corneum, was measured for each group of mice. As a result, while UV irradiation significantly decreased skin flexibility, the group treated with a substance that promotes the self-renewal ability of epidermal stem cells showed a significant improvement in skin flexibility compared to the control group treated with retinol (Figure 26). This revealed that substances that promote the cell competitiveness of epidermal stem cells have the effect of improving wrinkles while maintaining a good condition of the stratum corneum, compared to retinol.

[0174] Example 16: Skin roughness improvement test (apocynin) Using a skin irritation model induced with a 10% sodium lauryl sulfate (SDS) solution (a standard test for chemical-induced skin damage), the effectiveness of a skin irritation prevention or improvement agent in improving skin irritation was evaluated by administering a substance (apocynin) that promotes the competitive self-renewal ability of epidermal stem cells.

[0175] After acclimatizing hairless mice HOS:HR-1 females (7 weeks old) to the experimental environment, they were divided into the following three groups to ensure nearly uniform body weight. • Group not treated with SDS (n=2) • SDS treatment + solvent coating group (n=4) • SDS treatment + apocynin 200 μM coating group (n=4)

[0176] On the back of hairless mice, cotton pads soaked with 500 μL of 10% SDS solution (dissolved in 70% ethanol) were applied for 30 minutes each on the 1st, 2nd, 3rd, and 4th days. Apocynin was applied to the back daily from the day before the test until the 4th day. On the 3rd day after the start of the test, for the mice in each group, TEWL was measured at specific sites on the back (two symmetric sites on the left and right from the center of the backbone) in the same manner as above. Also on the 8th day, images were taken using a high-resolution three-dimensional image analyzer PRIMOS-CR (manufactured by Canfield Scientific, Inc., Integral Co., Ltd.), and based on the obtained images, for two symmetric sites on the left and right (each 10 mm x 10 mm) from the same center of the backbone as the site where TEWL was measured, the degree of skin roughness was measured using Primos OMC3_22 software with height-direction parameters representing skin surface roughness called "Ra (arithmetic mean roughness)" and "Rz (ten-point mean roughness).

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

[0178] From the above results, a substance (apocynin) that promotes the self-renewal ability of epidermal stem cells was found to have a significant effect on preventing or improving skin roughness.

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

[0180] On days 1, 2, 3, and 4, cotton balls soaked in 500 μL of 10% SDS solution (dissolved in 70% ethanol) were applied to the backs of hairless mice for 30 minutes each day. Ebselen was applied to the backs daily from the day before the experiment until day 4. On day 8 of the experiment, TEWL was measured for each group of mice at specific locations on the back (two locations symmetrically on either side of the center of the spine) in the same manner as described above. Simultaneously, images were captured using a high-resolution three-dimensional image analyzer PRIMOS-CR (manufactured by Canfield Scientific, Inc., Inc., operated by Integral Co., Ltd.). Based on the obtained images, the degree of skin roughness was measured using the Primos OMC3_22 software at two locations (10 mm x 10 mm each) symmetrically on either side of the center of the spine, the same locations where TEWL was measured, using height-direction parameters called "Ra (arithmetic mean roughness)" and "Rz (ten-point mean roughness)" to represent surface roughness.

[0181] As shown in Figures 29 and 30, the SDS-treated control group showed a significant increase in TEWL, and the Ra and Rz values, which are indicators of skin roughness, also increased, indicating that skin roughness was significantly induced. On the other hand, mice treated with a substance that promotes the self-renewal ability of epidermal stem cells (ebselen) showed a significant decrease in TEWL on the back compared to the control group treated with the solvent, and the Ra and Rz values ​​on the surface of the back also decreased significantly.

[0182] Based on these results, a substance that promotes the self-renewal ability of epidermal stem cells (ebselen) was found to have a significant effect in preventing or improving rough skin.

[0183] Example 18: Human skin roughness improvement test In a skin irritation test using a 10% sodium lauryl sulfate (SDS) solution (a standard test for chemical-induced skin damage in humans), two subjects were administered substances that promote the self-renewal ability of epidermal stem cells (apocinin, ebselen, and celecoxib) to evaluate the skin irritation prevention or improvement effects of these agents.

[0184] Six 1.5 cm square areas were created on the inner side of the subject's forearm, and the following treatment was performed on each area. • Group without SDS treatment (treated with 70% ethanol) • SDS treatment + solvent (50% ethanol / PBS) coating group • SDS treatment + apocynin (200 μM) solution coating group • SDS treatment + ebselen (0.25%) solution coating group • SDS treatment + celecoxib (2%) solution coating group

[0185] Cotton soaked in 500 μL of 10% SDS solution (dissolved in 70% ethanol) was applied to the test area for 30 minutes each on day 0 and day 1. Apocynin was applied to the test area once daily from day 0 (before and after SDS treatment) until day 9. TEWL was measured for each test area before and after SDS treatment, and daily from day 1 to day 7. For each area, the value obtained by subtracting the TEWL value before SDS treatment from the TEWL value on the measurement day was defined as "ΔTEWL," and this value was used for evaluation. The test area was also photographed with a digital camera and a high-resolution three-dimensional image analyzer PRIMOS-CR (manufactured by Canfield Scientific, Inc., Inc., operated by Integral Co., Ltd.). For PRIMOS images, the degree of skin roughness was measured using height-direction parameters called "Ry (maximum height)" and "Rz (ten-point mean roughness)," which represent surface roughness, using Primos OMC3_22 software. For each body part, the values ​​obtained by subtracting the Rz or Ry value of the untreated control from the Rz or Ry value on the measurement day were defined as "ΔRz" and "ΔRy," and these values ​​were used for evaluation.

[0186] Figures 31-33 show the test results. As shown in Figure 31, redness and skin roughness were clearly observed in the control and treated areas after SDS treatment. On day 7, redness and skin roughness persisted in the area treated with the control solution, but a significant reduction was observed in the areas treated with apocynin, ebselen, and celecoxib. Furthermore, on day 16, pigment deposition was observed in the affected area of ​​the control, but almost no pigment deposition was observed in the area treated with apocynin. Also, as shown in Figure 32, the ΔTEWL value increased significantly due to skin roughness induced by SDS, but the ΔTEWL value decreased significantly with the application of apocynin, ebselen, and celecoxib. Furthermore, as shown in the results in Figure 33, analysis of skin texture based on high-resolution images of the skin surface revealed that skin roughness was significantly induced in the SDS-treated control, but it was significantly reduced with the application of apocynin, ebselen, and celecoxib. Furthermore, while the ΔRz and ΔRy values, which indicate roughness of the skin surface and are indicators of skin irritation, significantly increased in the SDS-treated control group, these ΔRz and ΔRy values ​​significantly decreased in the affected areas treated with apocynin, ebselen, and celecoxib. This suggests that skin irritation and hyperpigmentation significantly induced by SDS solution can be remarkably improved by substances that promote the competitive self-renewal ability of epidermal stem cells.

[0187] The results shown in Figures 31-33 indicate that substances that promote the competitive self-renewal ability of epidermal stem cells exhibit significant preventive or ameliorative effects on rough skin in humans, suggesting their effectiveness in preventing and treating skin damage caused by chemicals and other factors.

[0188] Manufacturing Example 1 (1) Production of Apocynum venetum extract The Apocynum venetum extract used was manufactured by Tokiwa Botanical Chemical Research Institute Co., Ltd. Dried leaves of Apocynum venetum were crushed, 60% ethanol was added to the crushed material, and after heating and refluxing, the extract was filtered. The extraction residue was heated again with 60% ethanol and refluxed, then filtered to obtain another extract. The first and second extracts were combined, concentrated under reduced pressure, the pH was adjusted to 3, and the mixture was stirred overnight. After filtering to remove insoluble matter, the resulting filtrate was passed through a synthetic adsorbent resin, washed with water, and the fraction removed with 70% ethanol was collected. This fraction was then concentrated under reduced pressure and spray-dried to obtain the Apocynum venetum extract.

[0189] (2) Mulberry (Morus alba) extract The mulberry extract used was manufactured by Tokiwa Botanical Chemical Research Institute Co., Ltd. 50% ethanol was added to mulberry (Morus alba) leaves, heated under reflux, and filtered to obtain the extract. After concentrating the extract under reduced pressure, an excipient was added, and the mixture was dried to obtain the mulberry extract.

[0190] (3) Production of Gymnema sylvestre extract The Gymnema extract used was manufactured by Tokiwa Botanical Chemical Research Institute Co., Ltd. The crushed Gymnema sylvestre leaves were heated and reflux-extracted with 70% ethanol, then filtered to obtain the extract. The resulting extract was concentrated under reduced pressure, followed by reduced-pressure drying to obtain the Gymnema extract.

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

[0192] (5) Milk thistle (Silybum marianum) extract The milk thistle extract used was manufactured by Tokiwa Botanical Chemical Research Institute Co., Ltd. The milk thistle (Silybum marianum) fruit (defatted residue) was heated and refluxed with 90-95% ethanol, filtered, and the extract was obtained. The extract was concentrated under reduced pressure, purified with ethanol, then concentrated under reduced pressure and dried with warm air to obtain the milk thistle extract.

[0193] (6) Loquat (Eriobotrya japonica) leaf extract The loquat leaf extract used was manufactured by Tokiwa Botanical Chemical Research Institute Co., Ltd. The crushed loquat (Eriobotrya japonica) leaves were heated and reflux-extracted with 80% ethanol, and the extract was filtered. Activated carbon was added to the resulting extract for decolorization. The decolorized solution was then concentrated under reduced pressure and dried under reduced pressure to obtain the loquat leaf extract.

[0194] (7) Production of black turmeric (Kaempferia parviflora) extract The black turmeric extract used was manufactured by Tokiwa Botanical Chemical Research Institute Co., Ltd. Dried chips of black turmeric (Kaempferia parviflora) rhizomes were crushed in a mixer, 80% ethanol was added to the crushed material, and after heating under reflux, the extract was filtered. The extraction residue was again heated under reflux with 80% ethanol, filtered, and an extract was obtained. The first and second extracts were combined, concentrated under reduced pressure, an excipient was added, and the mixture was dried under reduced pressure to obtain the black turmeric extract.

[0195] Manufacturing Example 2 The plant extracts shown in Table 4 were extracted from various plant parts using predetermined extraction solvents.

[0196] [Table 4]

[0197] While preferred embodiments of the invention are shown herein, it will be apparent to those skilled in the art that such embodiments are provided for illustrative purposes only, and that they can be modified, altered, and substituted in various ways without departing from the invention. It should be understood that various alternative embodiments of the invention described herein can be used when carrying out the invention. Furthermore, the contents of all publications, including patents and patent applications referenced herein, should be construed as being incorporated herein by reference in the same way as the contents expressed herein. This application claims priority to Japanese Patent Application No. 2019-59616 (filed March 27, 2019), which is incorporated herein by reference in its entirety. [Industrial applicability]

[0198] As described above, the inventors have discovered 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 compositions useful for promoting skin wound healing, suppressing skin aging, controlling cell competition, and improving the regenerative capacity of epidermal stem cells. Continuing treatment due to skin damage caused by anticancer drugs is a major challenge and a problem affecting prognosis. The number of cancer patients who continue working while undergoing cancer treatment is increasing year by year, and the need for appearance care is extremely high. The compositions, cells, and methods disclosed herein can be effectively utilized in fields such as continuing anticancer drug treatment, surgical medicine, regenerative medicine, and cosmetic medicine.

Claims

1. A composition for suppressing blemishes or hyperpigmentation, characterized in that it contains apocynin as an active ingredient.

2. The composition according to claim 1, wherein the blemishes or pigmentation are due to pigment abnormalities caused by anticancer drugs, ultraviolet light, radiation, and / or oxidative stress.

3. A pharmaceutical composition, as described in claim 1 or 2.

4. A composition according to any one of claims 1 to 3, comprising a pharmaceutically acceptable excipient.

5. A composition according to any one of claims 1 to 4, for oral administration.

6. A composition according to any one of claims 1 to 4, which is a coating agent.

7. The composition according to claim 1 or 2, which is a cosmetic composition.

8. The composition according to claim 7, which is a composition for use in skincare.

9. The composition according to claim 7 or 8, further comprising at least one of a skin anti-aging agent, a skin tone enhancer, an anti-inflammatory agent, and a sunscreen.

10. A composition according to claim 1 or 2, which is a beauty supplement.

11. The composition according to claim 10, which is intended for oral ingestion.

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