Screening method for skin disease inhibitor

Abstract

To provide a screening method for novel skin disease inhibitors.SOLUTION: A comparison is made between gene expression 1 in human epidermal keratinocytes after a first culture step of culturing the human epidermal keratinocytes in the presence of Corynebacterium bacteria and gene expression 2 in human epidermal keratinocytes after a second culture step of culturing the human epidermal keratinocytes in the presence of Corynebacterium bacteria and the test substance. The comparison enables evaluation of the skin disease inhibitory effect of the test substance in the human epidermal keratinocytes.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for screening for a skin disease-suppressing agent. [Background technology]

[0002] Diseases of the epidermis and mucous membranes cause aesthetic problems and significantly reduce quality of life (QOL). These diseases include skin diseases such as atopic dermatitis, but the pathology of these skin diseases has not yet been fully elucidated, and no evidence-based treatment has been established.

[0003] It has been known that Staphylococcus aureus is involved in skin diseases such as atopic dermatitis (Non-Patent Document 1).

[0004] Experiments using mouse models are widely used to study the relationship between resident bacteria and epidermal and mucosal diseases. Since the skin is composed of various cells, including not only epidermal keratinocytes but also immune cells, it is thought that complex interactions occur between these cells. Therefore, the results obtained from experiments using model mice contain complex interactions, and care must be taken when interpreting the results. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Tetsuro Kobayashi et. al.,"Dysbiosis and Staphylococcus aureus colonization drives inflammation in atopic dermatitis" Immunity, 2015 April 21; 42(4) ;756-766 Summary of the Invention [Problem to be solved by the invention]

[0006] Under these circumstances, there is a demand for the development of new drugs to suppress skin diseases.

[0007] An object of one aspect of the present invention is to provide a method for screening for a new skin disease inhibitor. [Means for solving the problem]

[0008] Epidermal keratinocytes are cells that form the outermost barrier layer of the epithelium, thereby forming a mechanical defense mechanism in the skin, and also contribute to immune responses by secreting cytokines. The present inventors used epidermal keratinocytes as a simplified model of biological tissues to attempt to elucidate the interactions between resident bacteria and biological tissues.

[0009] The present inventors have found that when epidermal keratinocytes are cultured in the presence of bacteria of the genus Corynebacterium, the expression level of a gene associated with a skin disease changes, in other words, that a skin disease inhibitor can be screened by using the gene expressed in epidermal keratinocytes as an indicator, and have thus completed the present invention.

[0010] [1] A screening method for a skin disease-suppressing agent, comprising: a culture step 1 of culturing epidermal keratinocytes in the presence of Corynebacterium bacteria; a culture step 2 of culturing epidermal keratinocytes in the presence of Corynebacterium bacteria and a test substance; a measurement step of measuring gene expression 1 in the epidermal keratinocytes after culture step 1 and gene expression 2 in the epidermal keratinocytes after culture step 2; a comparison step of comparing gene expression 1 with gene expression 2; and an evaluation step of evaluating the effect of the test substance on suppressing a skin disease in the epidermal keratinocytes based on the results of the comparison step.

[0011] [2] The screening method described in [1], wherein the Corynebacterium bacteria used in the culture step 1 and the culture step 2 are killed Corynebacterium bacteria.

[0012] [3] The screening method according to [1] or [2], wherein the Corynebacterium bacterium is Corynebacterium durum and / or Corynebacterium matruchotii.

[0013] [4] The screening method described in any one of [1] to [3], wherein the gene expression 1 and the gene expression 2 measured in the measurement step are the expression of at least one gene selected from the group consisting of Keratin1, Keratin4, Keratin13, Keratin23, Keratin24, IL36G, CXCL8, IL33, and IL24. Effect of the Invention

[0014] According to one aspect of the present invention, a method for screening a new skin disease inhibitor can be provided. [Brief description of the drawings]

[0015] [Figure 1] 1 is a graph showing the results of a toxicity test of Corynebacterium bacteria against epidermal keratinocytes in an example of the present invention. [Diagram 2] 1 is a graph showing that Corynebacterium bacteria induce the expression of specific genes in epidermal keratinocytes in an example of the present invention. [Diagram 3] 1 is a graph showing that Corynebacterium bacteria induce the expression of specific genes in epidermal keratinocytes in an example of the present invention. [Figure 4] 1 is an image showing that Corynebacterium bacteria induce the expression of a specific gene in epidermal keratinocytes in an example of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] An example of an embodiment of the present invention will be described in detail below, but the present invention is not limited thereto. Various modifications of the present invention are possible within the scope of the claims. In addition, embodiments or examples obtained by appropriately combining the technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention. Furthermore, new technical features can be formed by combining the technical means disclosed in each embodiment. All academic documents and patent documents described in this specification are incorporated herein by reference. Unless otherwise specified in this specification, "X to Y" representing a numerical range is intended to mean "X or more and Y or less."

[0017] 1. Screening method for skin disease inhibitors A screening method for a skin disease inhibitor according to one embodiment of the present invention includes a culture step 1 of culturing epidermal keratinocytes in the presence of Corynebacterium bacteria, a culture step 2 of culturing epidermal keratinocytes in the presence of Corynebacterium bacteria and a test substance, a measurement step of measuring gene expression 1 in the epidermal keratinocytes after culture step 1 and gene expression 2 in the epidermal keratinocytes after culture step 2, a comparison step of comparing gene expression 1 with gene expression 2, and an evaluation step of evaluating the effect of the test substance on suppressing a skin disease in the epidermal keratinocytes based on the results of the comparison step.

[0018] A screening method for a skin disease inhibitor according to one embodiment of the present invention includes a culture step 1 in which epidermal keratinocytes are cultured in the presence of Corynebacterium bacteria, and a culture step 2 in which epidermal keratinocytes are cultured in the presence of Corynebacterium bacteria and a test substance.

[0019] The skin disease is not limited to, and may include, for example, keratinization abnormalities (e.g., mucocutaneous diseases caused by keratinization abnormalities) and immune-related diseases (e.g., inflammation). More specifically, the skin disease may include exfoliative cheilitis, atopic dermatitis, psoriasis, punctate keratolysis, acne, yellow fungus hair, erythrasma, periodontitis, oral squamous cell carcinoma, ichthyosis, palmoplantar keratosis, Darier's disease, lichen planus, and white sponge nevus.

[0020] In this specification, the term "suppressant" refers to a drug that provides a therapeutic effect and / or a preventive effect. The therapeutic effect refers to the effects exemplified in the following (1) to (3), and the preventive effect refers to the effects exemplified in the following (4) to (6), but is not limited to these effects.

[0021] (1) Reduce the severity of one or more symptoms of the disease compared to when the inhibitor is not administered: (2) Prevent an increase in the severity of one or more symptoms of the disease compared to when the inhibitor is not administered: (3) reducing the rate at which the severity of one or more symptoms associated with the disease increases compared to when the inhibitor is not administered: (4) Preventing the onset of, or reducing the risk of onset of, one or more symptoms of a disease compared to not administering the inhibitor: (5) Preventing or reducing the risk of recurrence of one or more symptoms of a disease compared to not administering the inhibitor: (6) Preventing the onset of, or reducing the risk of the onset of, one or more symptoms of a disease compared to when the inhibitor is not administered.

[0022] The epidermal keratinocytes are not limited, and may be epidermal keratinocytes collected from a living body, non-human epidermal keratinocytes, or established epidermal keratinocytes (e.g., NHEK, HaCaT). The origin of the epidermal keratinocytes is not limited, and the epidermal keratinocytes may be human epidermal keratinocytes or non-human epidermal keratinocytes. The non-human keratinocytes may be cells derived from, for example, monkeys, chimpanzees, cows, pigs, sheep, goats, horses, dogs, cats, rabbits, mice, rats, or hamsters.

[0023] The culture form in the culture step 1 and the culture step 2 is not limited, and examples thereof include (i) a form in which Corynebacterium bacteria or a part of Corynebacterium bacteria is added to a medium in which epidermal keratinocytes are cultured, and then the epidermal keratinocytes are cultured, (ii) a form in which Corynebacterium bacteria or a medium containing a part of Corynebacterium bacteria is added to epidermal keratinocytes, and then the epidermal keratinocytes are cultured, or (iii) a form in which Corynebacterium bacteria or a medium containing a part of Corynebacterium bacteria is added to a medium in which epidermal keratinocytes are cultured, and then the epidermal keratinocytes are cultured. The medium is not limited, and a known medium used for culturing epidermal keratinocytes can be used. Various conditions during culture (e.g., temperature, CO 2 The concentration of the culture medium is not limited, and known conditions used for culturing epidermal keratinocytes can be used.

[0024] The Corynebacterium bacteria used in the culture step 1 and the culture step 2 may be killed bacteria of the Corynebacterium genus or may be live bacteria of the Corynebacterium genus. From the viewpoint of preventing aggregation and making it easy to control the amount of bacteria, the Corynebacterium bacteria used in the culture step 1 and the culture step 2 are preferably killed bacteria of the Corynebacterium genus.

[0025] An example of a method for preparing killed bacteria of the genus Corynebacterium is a method of treating the bacteria at high temperature. More specifically, an example of a method for preparing killed bacteria of the genus Corynebacterium is a method of treating the bacteria at high temperature (e.g., 80°C to 100°C) for a predetermined time (e.g., 15 minutes to 1 hour). Examples of killed bacteria include (i) bacteria of the genus Corynebacterium treated at high temperature, (ii) a desired purified fraction obtained by treating bacteria of the genus Corynebacterium at high temperature to obtain bacteria, and then further purifying the bacteria, (iii) a solution containing bacteria of the genus Corynebacterium treated at high temperature, and (iv) a desired purified fraction obtained by treating a solution containing bacteria of the genus Corynebacterium at high temperature to obtain a treated solution, and then further purifying the treated solution.

[0026] The bacterium of the genus Corynebacterium is not limited, but is preferably Corynebacterium durum and / or Corynebacterium matruchotii. With this configuration, skin disease inhibitors can be efficiently screened. These bacteria can be obtained, for example, from the RIKEN BioResource Research Center.

[0027] The culture time in the culture step 1 and the culture step 2 is not limited and may be, for example, 1 hour to 100 hours, 1 hour to 90 hours, or 1 hour to 80 hours. From the viewpoint of contacting epidermal keratinocytes with the Corynebacterium bacteria for a long period of time while suppressing unnecessary effects of the Corynebacterium bacteria on epidermal keratinocytes (for example, effects of inhibiting the proliferation of epidermal keratinocytes, effects of killing epidermal keratinocytes, etc.), the culture time in the culture step 1 and the culture step 2 is preferably 1 hour to 80 hours, more preferably 3 hours to 80 hours, and more preferably 6 hours to 72 hours.

[0028] The test substance is not limited, and may be, for example, an inorganic compound, an organic compound, a plant extract, a microbial extract, or a culture supernatant of various cells. The test substance may be used as it is, or may be used in a state dissolved in a solvent. Examples of the solvent include physiological saline, phosphate buffered saline, and water.

[0029] A screening method for a skin disease inhibitor according to one embodiment of the present invention includes a measurement step of measuring gene expression 1 in epidermal keratinocytes after a culture step 1 and gene expression 2 in epidermal keratinocytes after a culture step 2.

[0030] The gene expression 1 and gene expression 2 measured in the measuring step are not limited, but are preferably the expression of at least one gene (1, 2, 3, 4, 5, 6, 7, 8, or 9) selected from the group consisting of Keratin1, Keratin4, Keratin13, Keratin23, Keratin24, IL36G, CXCL8, IL33, and IL24. With this configuration, skin disease inhibitors can be efficiently screened.

[0031] The expression levels of keratin genes such as Keratin1, Keratin4, Keratin13, Keratin23, and Keratin24 are increased in skin affected by skin diseases (e.g., keratinization abnormality, mucocutaneous diseases caused by keratinization abnormality). By using keratin genes such as Keratin1, Keratin4, Keratin13, Keratin23, and Keratin24, it is possible to more efficiently screen skin disease inhibitors. More specifically, by using keratin genes such as Keratin1, Keratin4, Keratin13, Keratin23, and Keratin24, it is possible to more efficiently screen inhibitors of exfoliative cheilitis, atopic dermatitis, psoriasis, punctate keratolysis, acne, yellow fungus hair, erythrasma, white sponge nevus, lichen planus, ichthyosis, palmoplantar keratosis, or Darier's disease.

[0032] Immune response molecule genes such as IL36G, CXCL8, IL33, and IL24 are highly expressed in immune-related cells and tissues. In skin affected by skin diseases (e.g., immune-related diseases, inflammation), these genes are expressed in greater amounts. By using immune response molecule genes such as IL36G, CXCL8, IL33, and IL24, skin disease inhibitors can be screened more efficiently. More specifically, by using immune response molecule genes such as IL36G, CXCL8, IL33, and IL24, inhibitors of atopic dermatitis, psoriasis, periodontitis, and oral squamous cell carcinoma can be screened more efficiently.

[0033] The gene expression 1 and gene expression 2 measured in the measurement step may be, for example, the expression level of a gene. The expression level of the gene may be the expression level of mRNA transcribed from the gene, or the expression level of a protein translated from the mRNA. That is, in the measurement step, either the expression level of mRNA derived from a marker gene and / or the expression level of a protein derived from a marker gene may be measured.

[0034] The method for measuring the expression level of mRNA and the expression level of protein is not limited, and known methods can be used. Examples of the method for measuring the expression level of mRNA include real-time RT-PCR, RNase protection assay, Northern blot analysis, dot blot, RNA-sequence analysis, microarray, and FISH (fluorescence in situ hybridization). On the other hand, examples of the method for measuring the expression level of protein include Western blot, immunoprecipitation, immunohistochemistry, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), EIA (enzyme immunoassay), bioassay, and flow cytometry.

[0035] For example, the cDNA nucleotide sequences and protein amino acid sequences of human Keratin4, Keratin13, Keratin23, Keratin24, IL36G, CXCL8, IL33, and IL24 are known.

[0036] The nucleotide sequence of human Keratin1 cDNA is registered under Accession No. NM_006121.4, and the amino acid sequence of human Keratin1 protein is registered under Accession No. NP_006112.3.

[0037] The nucleotide sequence of human Keratin4 cDNA is registered under Accession No. NM_002272.4, and the amino acid sequence of human Keratin4 protein is registered under Accession No. NP_002263.3.

[0038] The nucleotide sequence of human Keratin 13 cDNA is registered under Accession No. NM_153490.3, and the amino acid sequence of human Keratin 13 protein is registered under Accession No. NP_705694.3.

[0039] The nucleotide sequence of human Keratin 23 cDNA is registered under Accession No. NM_015515.5, and the amino acid sequence of human Keratin 23 protein is registered under Accession No. NP_056330.3.

[0040] The nucleotide sequence of human Keratin24 cDNA is registered under Accession No. NM_019016.3, and the amino acid sequence of human Keratin24 protein is registered under Accession No. NP_061889.2.

[0041] The nucleotide sequence of human IL36G cDNA has been registered under Accession No. NM_019618.4, and the amino acid sequence of human IL36G protein has been registered under Accession No. NP_062564.1.

[0042] The nucleotide sequence of human CXCL8 cDNA has been registered under Accession No. NM_000584.4, and the amino acid sequence of human CXCL8 protein has been registered under Accession No. NP_000575.1.

[0043] The nucleotide sequence of human IL33 cDNA is registered under Accession No. NM_033439.4, and the amino acid sequence of human IL33 protein is registered under Accession No. NP_254274.1.

[0044] The nucleotide sequence of human IL24 cDNA is registered under Accession No. NM_006850.3, and the amino acid sequence of human IL24 protein is registered under Accession No. NP_006841.1.

[0045] When measuring the expression level of mRNA, a probe or primer, etc., may be prepared according to a known method based on the information on the base sequence of the cDNA, and the expression level of mRNA may be measured according to the above-mentioned method. On the other hand, when measuring the expression level of a protein, an antibody, etc. may be prepared according to a known method based on the information on the amino acid sequence of the protein, and the expression level of the protein may be measured according to the above-mentioned method. Of course, for organisms other than humans, information on the base sequence of the cDNA of the gene and information on the amino acid sequence of the protein of the gene are publicly known, so that a probe, primer, antibody, etc. may be prepared based on this information.

[0046] A screening method for a skin disease inhibitor according to one embodiment of the present invention includes a comparison step of comparing gene expression 1 with gene expression 2, and an evaluation step of evaluating the effect of a test substance on inhibiting a skin disease in epidermal keratinocytes based on the results of the comparison step.

[0047] As an example, consider gene A, the expression level of which increases when epidermal keratinocytes are cultured in the presence of bacteria of the genus Corynebacterium. If gene expression 2 of gene A is less than gene expression 1 of gene A (gene expression 2<gene expression 1), the test substance can be evaluated as having an inhibitory effect on skin diseases.

[0048] As an example, consider gene B, the expression level of which decreases when epidermal keratinocytes are cultured in the presence of bacteria of the genus Corynebacterium. If gene expression 2 of gene B is greater than gene expression 1 of gene B (gene expression 2>gene expression 1), the test substance can be evaluated as having an inhibitory effect on skin diseases.

[0049] This invention may also contribute to achieving Goal 3 of the United Nations' Sustainable Development Goals (SDGs), which aims to "ensure good health and promote well-being for all." EXAMPLES

[0050] <1.Cell culture> Normal human epidermal keratinocytes (NHEK) were cultured in EpiLife® Medium (Thermo Fisher Scientific, MEPI500CA) containing 60 μM calcium and HuMedia-KG growth additive (Kurabo, model number: KK-6150) at 37°C and 5% CO. 2 The cells were cultured in an incubator under the following conditions.

[0051] <2. Bacterial culture> Staphylococcus epidermidis (NBRC100911) was obtained from the NITE Biotechnology Center. The Staphylococcus epidermidis was cultured on an agar medium prepared using Soybean-Casein Digest Agar DAIGO (Nihon Seiyaku 396-00175) at 37°C and 5% CO 2 The Staphylococcus epidermidis was cultured in an incubator under the conditions of 37°C and 170 rpm for 24 hours. After 24 hours from the start of the culture, the Staphylococcus epidermidis was inoculated into Soybean-Casein Digest Broth DAIGO (Nihon Seiyaku 393-00185). The Staphylococcus epidermidis was cultured with shaking for another 24 hours under the conditions of 37°C and 170 rpm for 24 hours, and then used in the experiment.

[0052] Corynebacterium durum (JCM11948) and Corynebacterium matruchotii (JCM9386) were obtained from the RIKEN BioResource Research Center. These bacteria were shake-cultured for 24 hours at 37°C and 170 rpm in Brain Heart Infusion Broth Nissui (Nissui Pharmaceutical, 05509) containing 2% Bacto (registered trademark) Yeast Extract (Thermo Fisher Scientific, 212750) and 1.5% Bacto (registered trademark) Agar (Becton Dickinson, 214010). An equal amount of medium was then added, and the bacteria were shake-cultured for another 24 hours before being used in the experiment.

[0053] <3. Co-culture> The bacteria cultured by the method described above in <2. Bacterial culture> were dispersed in PBS and then heated at 90° C. for 30 minutes to obtain killed bacteria.

[0054] For DNA microarray and immunocytostaining described below, protein concentration was measured and killed bacteria adjusted to a protein concentration of 100 μg / mL using phosphate-buffered saline (PBS) were used. For RT-qPCR described below, turbidity was measured and a concentration of 1.5 × 107 Killed bacteria adjusted to CFU / mL were used.

[0055] At the start of co-culture, the PBS in which the dead bacteria were dispersed was replaced with NHEK culture medium to prepare an NHEK culture medium in which the dead bacteria were dispersed. The NHEK culture medium was replaced with the medium in which NHEK, which had been cultured in advance by the method described above in <1. Cell culture>, and incubated at 37°C, 5% CO 2 NHEKs and bacteria were co-cultured in an incubator under the following conditions.

[0056] Co-culture was performed for 6, 24, 48, or 72 hours to determine the culture conditions (culture time) that would not cause toxicity to NHEK. The study was performed using the Cell counting kit-8 (DOJINDO), a cell proliferation / cytotoxicity assay kit, following the protocol attached to the kit.

[0057] The test results are shown in Figure 1. As is clear from Figure 1, the survival rate of NHEK was maintained at a high level in the co-culture for 6, 24, 48, and 72 hours, indicating that the co-culture for 6, 24, 48, and 72 hours was not toxic to NHEK. Therefore, these culture conditions were adopted in the subsequent tests.

[0058] 4. RNA Isolation NHEKs were washed with PBS, and RNA was obtained from the NHEKs using NucleoSpin (registered trademark) RNA Plus (MACHEREY-NAGEL, model number: 740984). The specific method for isolating RNA was in accordance with the protocol attached to the kit.

[0059] <5.DNA microarray> For microarray analysis, GeneChip® Human Genome U133 Plus 2.0 Array (Thermo Fisher Scientific, model number: 900467) was used.

[0060] Co-culture was performed according to the method described above in <3. Co-culture>. After 6, 24, or 72 hours of co-culture, RNA was isolated from each NHEK according to the method described above in <4. RNA isolation>.

[0061] cDNA was synthesized from the RNA. Labeling, fragmentation, and hybridization were performed using the GeneChip® 3' IVT PLUS Reagent Kit (Thermo Fisher Scientific, model number: 902416). Data analysis was performed using the Transcriptome Analysis Console software (Thermo Fisher Scientific).

[0062] The test results are shown in Table 1 below. The test results for Keratin 4, Keratin 13, Keratin 23, and Keratin 24 were obtained using RNA isolated from NHEKs after 72 hours of co-culture and RNA isolated from NHEKs not co-cultured.

[0063] IL36G, CXCL8, and IL33 results were obtained using RNA isolated from NHEKs after 6 hours of co-culture and from NHEKs not co-cultured.

[0064] The IL24 results were obtained using RNA isolated from NHEKs after 24 hours of co-culture and from NHEKs not co-cultured.

[0065] Table 1 shows the ratio of "expression level of a specific gene in NHEK after co-culture / expression level of a specific gene in NHEK without co-culture."

[0066] [Table 1]

[0067] As is clear from Table 1, the expression levels of Keratin4, Keratin13, Keratin23, Keratin24, IL36G, CXCL8, IL33, and IL24 were increased by Corynebacterium bacteria.

[0068] <6.RT-qPCR> Co-culture was performed according to the method described above in <3. Co-culture>. In the co-culture, Corynebacterium matruchotii (JCM9386) was used as the bacterium to be tested, and Staphylococcus epidermidis (NBRC100911) was used as the negative control bacterium. After co-culture for 6, 24, or 72 hours, RNA was isolated from each NHEK according to the method described above in <4. RNA isolation>.

[0069] cDNA was synthesized from the RNA using QuantiTect (registered trademark) Reverse Transcription Kit (200) (QIAGEN, model number: 205315). The gene sequence was specifically amplified using the cDMA and THUNDERBIRD (registered trademark) Next SYBR qPCR Mix (TOYOBO, model number: QPX-201). GAPDH RNA was used as an endogenous control during detection. The primers used were those shown in Table 2 below.

[0070] [Table 2]

[0071] The PCR reaction was performed using a QuantStudio 6 Pro (Thermo Fisher Scientific) and consisted of 40 reaction cycles consisting of a denaturation reaction at 95°C for 15 seconds, an annealing reaction at 55°C for 10 seconds, and an extension reaction at 72°C for 20 seconds.

[0072] The test results are shown in Figure 2. Figure 2 shows how the expression level of each gene changed after 72 hours of co-culture, based on the expression level of each gene after 6 hours of co-culture.

[0073] As is clear from FIG. 2, the expression levels of Keratin 4, Keratin 13, and Keratin 24 were increased by Corynebacterium bacteria.

[0074] Another test result is shown in Figure 3. Figure 3 shows how the expression level of each gene changed when co-culture was performed for 6 hours, 24 hours, or 72 hours, based on the expression level of each gene when co-culture was not performed. Note that statistical analysis for Figure 3 was performed using GraphPad Prism version 9.5.1. Significance tests were performed using One-way ANOVA and Fisher's least significant difference test.

[0075] As is clear from FIG. 3, the expression levels of Keratin1, Keratin13, Keratin24, IL36G, CXCL8, IL33, and IL24 were increased by Corynebacterium bacteria.

[0076] <7. Immune cell staining> Co-culture was performed for 72 hours according to the method described above in <3. Co-culture>. After co-culture, the NHEKs were fixed overnight at 4°C using 4% paraformaldehyde phosphate buffer (Fujifilm Wako Pure Chemical Industries, model number: 163-20145).

[0077] The fixed NHEK was washed three times with PBS, and then incubated in a blocking solution of 5% FBS (fetal bovine serum) (Thermo Fisher Scientific, model number: 10270-106) / 0.1% TritonX-100 (Sigma, model number: T8787) at room temperature for 30 minutes for blocking. The NHEK was then reacted with a primary antibody overnight at 4°C. The primary antibodies used and the dilution ratios are shown in Table 3 below.

[0078] [Table 3]

[0079] After reacting with the primary antibody, the NHEKs were washed in phosphate-buffered saline (PBST) containing 0.1% Tween-20 (Sigma, model number: P9416) and then reacted with the secondary antibody at room temperature for 2 hours in the dark. The secondary antibody used was an anti-rabbit IgG donkey polyclonal antibody (1:200) (Thermo Fisher Scientific, model number: A-21207). Nuclear staining was performed using Hoechst 33342 (1:1000) (Thermo Fisher Scientific, model number: H3570).

[0080] After reacting with the secondary antibody and Hoechst 33342, the NHEKs were washed with PBST and then sealed in a cover glass using FluorSave (registered trademark) Reagent (Merck, model number: 345789). The NHEKs were observed using a confocal laser scanning microscope (OLYMPUS, product name: FV3000 IX83).

[0081] The test results are shown in Figure 3. In Figure 3, stained images of Keratin 13 were observed in the cells indicated by the arrows. As is clear from Figure 3, the expression level of Keratin 13 was increased by the Corynebacterium bacteria. [Industrial Applicability]

[0082] The present invention can be used for screening of skin disease inhibitors. More specifically, the present invention can be suitably used for the development of cosmetics, medicines, or quasi-drugs.

Claims

1. A culture step 1 of culturing epidermal keratinocytes in the presence of bacteria of the genus Corynebacterium; a culturing step 2 of culturing epidermal keratinocytes in the presence of Corynebacterium sp. and a test substance; a measuring step of measuring gene expression 1 in the epidermal keratinocytes after the culture step 1 and gene expression 2 in the epidermal keratinocytes after the culture step 2; A comparison step of comparing the gene expression 1 with the gene expression 2; and A screening method for a skin disease inhibitor, comprising an evaluation step of evaluating the effect of the test substance on inhibiting a skin disease in epidermal keratinocytes based on the results of the comparison step.

2. The screening method according to claim 1 , wherein the Corynebacterium bacterium used in the culture step 1 and the culture step 2 is a killed Corynebacterium bacterium.

3. The method for screening according to claim 1 or 2, wherein the bacterium of the genus Corynebacterium is Corynebacterium durum and / or Corynebacterium matruchotii.

4. The screening method according to claim 1, wherein the gene expression 1 and the gene expression 2 measured in the measurement step are the expression of at least one gene selected from the group consisting of Keratin 1, Keratin 4, Keratin 13, Keratin 23, Keratin 24, IL36G, CXCL8, IL33, and IL24.

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