Method for evaluating therapeutic and / or preventive action on skin diseases caused by obstruction and / or inflammation of pilosebaceous unit, and method for searching therapeutic and / or preventive target on skin diseases caused by obstruction and / or inflammation of pilosebaceous unit

Abstract

To provide a method for evaluating the therapeutic and / or prophylactic action of a skin disease caused by the obstruction and / or inflammation of a pilosebaceous gland system.SOLUTION: A method for evaluating a therapeutic and / or prophylactic action of a substance or agent on a skin disease, the method comprising: bringing the substance or agent into contact with sebocytes or progenitor cells thereof; and evaluating the sebocytes or progenitor cells thereof, wherein the skin disease is a skin disease caused by obstruction and / or inflammation of the pilosebaceous unit.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a method for evaluating the therapeutic and / or preventive effect of a skin disease caused by obstruction and / or inflammation of the pilosebaceous system, and a method for searching for therapeutic and / or preventive targets for a skin disease caused by obstruction and / or inflammation of the pilosebaceous system. [Background technology]

[0002] In skin diseases caused by obstruction and / or inflammation of the pilosebaceous system, such as acne vulgaris (hereinafter also referred to as "pilosebaceous skin diseases"), symptoms and / or pathologies such as comedones, papules, pustules, nodules, and cysts occur as a result of obstruction and inflammation of the pilosebaceous system (hair follicles and their associated sebaceous glands). Regarding skin diseases and lipids, for example, Non-Patent Document 1 discloses that in inflammatory skin diseases that have already developed, differentiation of macrophages into TREM2 macrophages after contact with squalene reduces the bactericidal ability of Cutibacterium acnes, which may exacerbate inflammation. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Tran H. Do et al., "TREM2macrophages induced by human lipids drive inflammation in acne lesions." Sci.Immunol. 7, eabo2787 (2022). [Non-patent document 2] Simon Joost et al., "Single-CellTranscriptomics Reveals that Differentiation and Spatial Signatures ShapeEpidermal and Hair Follicle Heterogeneity", Cell Systems, Volume 3, Issue3, Pages 221-237.e9, (2016). [Non-patent document 3] Tetsuro Kobayashi et al.,"Homeostatic Control of Sebaceous Glands by Innate Lymphoid CellsRegulates Commensal Bacteria Equilibrium", Cell, Volume 176, Issue 5, 2019,Pages 982-997. Summary of the Invention [Problem to be solved by the invention]

[0004] Regarding pilosebaceous gland-derived skin diseases, the process of their onset is largely unknown compared to the process of disease deterioration after the onset. Therefore, there are few reported examples or methodologies that have evaluated the therapeutic and / or preventive effects of substances or agents on pilosebaceous gland-derived skin diseases or explored further therapeutic and / or preventive targets for pilosebaceous gland-derived skin diseases, starting from the mechanism of onset of pilosebaceous gland-derived skin diseases.

[0005] The present disclosure aims to provide a method for evaluating the therapeutic and / or preventive effect of a substance or agent on a pilosebaceous gland-derived skin disease. The present disclosure also aims to provide a method for screening a substance or agent that prevents and / or treats a pilosebaceous gland-derived skin disease. A further object of the present disclosure is to provide a method for searching for therapeutic and / or preventive targets for a pilosebaceous gland-derived skin disease. [Means for solving the problem]

[0006] The present inventors have investigated the onset process of pilosebaceous gland-derived skin diseases using a skin disease model mouse that they independently developed. As a result, the present inventors have found that, in the early stage of the onset of pilosebaceous gland-derived skin diseases, keratinization and / or inflammatory signaling are particularly enhanced in sebocytes or their precursor cells, and that, as a result, tests using sebocytes or their precursor cells can be used to evaluate the therapeutic and / or preventive effects of substances or agents on pilosebaceous gland-derived skin diseases and to explore further therapeutic and / or preventive targets for pilosebaceous gland-derived skin diseases from a perspective different from that of conventional methodologies.

[0007] The present disclosure relates, for example, to the following: [1] A method for evaluating the therapeutic and / or preventive effect of a substance or agent on a skin disease, comprising: contacting a substance or agent with sebocytes or their precursor cells; and evaluating the sebocytes or their precursor cells; The method, wherein the skin disease is a skin disease resulting from blockage and / or inflammation of the pilosebaceous system. [2] A method for screening a substance or agent that prevents and / or treats a skin disease, comprising: contacting a candidate substance or agent with a sebocyte or a precursor cell thereof; and evaluating the sebocytes or their precursor cells; The method, wherein the skin disease is a skin disease resulting from blockage and / or inflammation of the pilosebaceous system. [3] A method for exploring targets for treating and / or preventing skin diseases, comprising: The method comprises artificially increasing, decreasing or inhibiting the amount or activity of a protein that is a candidate target for the treatment and / or prevention of the above-mentioned skin disease, in a cell or animal having a sebocyte or a precursor cell thereof, wherein the target cell is a sebocyte or a precursor cell thereof, The method, wherein the skin disease is a skin disease resulting from blockage and / or inflammation of the pilosebaceous system. [4] The method according to any one of [1] to [3], wherein the sebocytes or precursor cells thereof are cells present in an animal. [5] The method according to any one of [1] to [3], wherein the sebocytes or their precursor cells are isolated cells. [6] The method according to any one of [1] to [5], wherein the sebaceous gland cells or precursor cells thereof are sebaceous gland precursor cells. [7] The method according to any one of [1] to [6], wherein the sebocytes or precursor cells thereof are Lrig1-positive cells. [8] The method according to any one of [1] to [7], wherein the skin disease is an inflammatory skin disease caused by hyperkeratosis. [9] The method according to any one of [1] to [8], wherein the skin disease is acne vulgaris.

[10] A method according to any one of [1] to [9], wherein evaluating the sebaceous gland cells or their precursor cells comprises evaluating the amount of at least one selected from the group consisting of genes, proteins and lipids in the sebaceous gland cells or their precursor cells.

[11] The method according to any one of [1] to

[10] , wherein evaluating the sebaceous gland cells or their precursor cells comprises evaluating the amount of a keratinization marker and / or a chemokine-related marker in the sebaceous gland cells or their precursor cells.

[12] The method according to

[11] , wherein the keratinization marker includes Krt17.

[13] The method according to

[11] or

[12] , wherein the chemokine-related marker includes Cxcl1.

[14] The method according to any one of [1] to

[13] , further comprising contacting the sebocytes or their precursor cells with a C14 to C18 fatty acid and / or an alkyl ester thereof.

[15] The method according to

[14] , wherein the C14 to C18 fatty acid is palmitic acid.

[16] The method according to

[14] or

[15] , wherein the alkyl ester is an ethyl ester. [Effects of the Invention]

[0008] The present disclosure provides a method for evaluating the therapeutic and / or preventive effect of a substance or agent on a pilosebaceous gland-derived skin disease. The present disclosure also provides a method for screening a substance or agent that prevents and / or treats a pilosebaceous gland-derived skin disease. Furthermore, the present disclosure provides a method for exploring targets for the treatment and / or prevention of a pilosebaceous gland-derived skin disease.

[0009] The evaluation method, screening method, and exploration method according to the present disclosure evaluate sebocytes or their precursor cells, which show particularly enhanced keratinization and / or inflammatory signaling in the early stage of the onset of pilosebaceous gland-derived skin diseases. Therefore, the evaluation method, screening method, and exploration method according to the present disclosure are suitable for evaluating the therapeutic and / or preventive effects of substances or agents that effectively act on the onset process of pilosebaceous gland-derived skin diseases, and for exploring further therapeutic and / or preventive targets for pilosebaceous gland-derived skin diseases. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows an image of a hematoxylin-eosin stained section prepared using tissue collected from a model mouse in Reference Example 1. [Figure 2] FIG. 1 shows the results of measuring the cross-sectional area of ​​hair follicles in a model mouse in a direction perpendicular to the epidermis in Reference Example 1. [Figure 3] FIG. 1 shows the results of quantifying inflammatory markers (a) Il-1α, (b) Il-1β, (c) Tnf, and (d) Defb4 contained in tissues collected from mice in Reference Example 1. [Figure 4] FIG. 1 shows the results of quantifying inflammatory markers (a) Defb14, (b) S100a8, (c) S100a9, and (d) Tslp contained in tissues collected from mice in Reference Example 1. [Figure 5] FIG. 1 shows the results of quantifying hyperkeratosis markers (a) Krt16, (b) Klk6, and (c) Krt17 contained in tissues collected from mice in Reference Example 1. [Figure 6]FIG. 1 is a diagram in which the results of single-cell RNA-seq analysis in Example 1 are plotted by reducing the dimension of the expression profile using Uniform Manifold Approximation and Projection (UMAP). [Figure 7] FIG. 7 shows the expression levels of various markers used to identify the origin of cells in mouse skin tissue in the cells plotted in FIG. 6 in Example 1. [Figure 8] FIG. 7 is a diagram visualizing the expression levels of various markers used to identify the origin of cells in mouse skin tissue for each classified cluster in the cells plotted in FIG. 6 in Example 1. [Figure 9] FIG. 7 shows plots for the control, 6 hours after application, and 12 hours after application, which are the basis for FIG. 6, in Example 1. [Figure 10] This figure shows the expression levels of various markers in four cell populations (4_0 to 4_3) further classified within the cluster classified as cells derived from hair follicle keratinocytes, and in three cell populations (10_0 to 10_2) further classified within the cluster classified as cells derived from sebaceous glands, as determined by single-cell RNA-seq analysis in Example 1. [Figure 11] FIG. 11 shows the expression levels of various markers in the two Lrig1-positive cell populations found in FIG. 10 in Example 1, compared with the expression levels in epidermal keratinocytes. DETAILED DESCRIPTION OF THE INVENTION

[0011] Although the embodiments for carrying out the present disclosure will be described, the present disclosure should not be construed as being limited to the following embodiments.

[0012] Sebaceous gland cells are mature cells that form the sebaceous glands within hair follicles. Sebaceous gland cells have the function of secreting lipids (sebum). Marker factors for sebaceous gland cells include Scd1 (Stearoyl-CoA Desaturase 1) and Mgst1 (Microsomal Glutathione S-Transferase 1). Sebaceous gland progenitor cells are immature cells that do not differentiate into sebaceous gland cells. Marker factors for sebaceous gland progenitor cells include Cd34 (Cluster of Differentiation 34) and Lrig1 (Leucine-rich repeats and immunoglobulin-like domains 1).

[0013] The present inventors have found that, among sebaceous gland cells and their precursor cells, Lrig1-positive cells are prone to elevated inflammatory markers in the early stages of pilosebaceous gland-derived skin diseases, and may be involved in the onset of pilosebaceous gland-derived skin diseases. The Lrig1 protein is present on the cell surface and has functions such as regulating the differentiation of undifferentiated cells. It has also been reported that, in hair follicles, Lrig1 is abundant in undifferentiated cells in the infundibulum, and is particularly expressed characteristically in sebaceous gland precursor cells present in the infundibulum (Non-Patent Document 2). Thus, the sebaceous gland cells and their precursor cells according to the present disclosure may, in a preferred embodiment, be cells present in or derived from the infundibulum, or in another preferred embodiment, be sebaceous gland precursor cells, or in a more preferred embodiment, be Lrig1-positive cells, or in an even more preferred embodiment, be Lrig1-positive sebaceous gland precursor cells. Cells present in or derived from the infundibulum of the hair follicle and sebaceous progenitor cells are preferred because they contain abundant Lrig1-positive cells. Whether a certain sebaceous gland cell or its precursor cell is derived from the infundibulum of the hair follicle can be evaluated by single-cell transcriptome analysis, for example, as described in Non-Patent Document 2.

[0014] Lrig1-positive cells may be cells in which mRNA encoding Lrig1 is significantly detected in an RNA-containing sample prepared from the cells. More specifically, Lrig1-positive cells may be cells in which mRNA encoding Lrig1 is significantly detected, for example, by measurement using a next-generation sequencer, a microarray, or reverse transcription-PCR. Furthermore, Lrig1-positive cells may be cells in which the amount of mRNA measured by these methods is significantly or significantly higher than the amount of mRNA similarly measured in Lrig1-negative cells derived from the same organism, for example, cells in which the amount of mRNA encoding Lrig1 is significantly or significantly higher than the amount of mRNA encoding Lrig1 in epidermal keratinocytes derived from the same organism.

[0015] Skin diseases caused by obstruction and / or inflammation of the pilosebaceous system (also referred to as "pilosebaceous-derived skin diseases" in the present disclosure) are diseases accompanied by symptoms and / or pathologies resulting from obstruction and inflammation of the pilosebaceous system (hair follicles and associated sebaceous glands). These diseases cause symptoms and / or pathologies, such as the formation of comedones, papules, pustules, nodules, cysts, etc., due to the obstruction and inflammation of the pilosebaceous system. The obstruction of the pilosebaceous system may be, for example, obstruction by lipids, or may be obstruction by lipids secreted by the pilosebaceous glands. The inflammation of the pilosebaceous system may be, for example, inflammation caused by exposure of the pilosebaceous system to lipids, or may be inflammation caused by exposure of the pilosebaceous system to lipids secreted by the pilosebaceous glands. The obstruction and / or inflammation of the pilosebaceous system may occur, for example, in the sebaceous gland and / or upper part of the hair follicle, around the infundibulum of the sebaceous gland and / or upper part of the hair follicle, or may occur in the infundibulum.

[0016] The pilosebaceous gland-derived skin disease may be, for example, inflammatory. The pilosebaceous gland-derived skin disease may be a skin disease derived from hyperkeratinization. A skin disease derived from hyperkeratinization is a skin disease caused by hyperkeratinization, which is an abnormality in keratinization in the epidermis. When hyperkeratinization occurs, hyperkeratinization occurs, which causes hair follicles to expand and accumulate sebum, resulting in various skin diseases. Furthermore, hair follicles that accumulate sebum are prone to inflammation. In a preferred embodiment, the pilosebaceous gland-derived skin disease may be an inflammatory skin disease derived from hyperkeratinization.

[0017] The pilosebaceous gland-derived skin disease may be, for example, keratosis, inflammatory keratosis, acne vulgaris, folliculitis, seborrheic dermatitis, hidradenitis suppurativa, or eczema, and in a preferred embodiment, may be acne vulgaris, all of which are skin diseases derived from hyperkeratinization.

[0018] A first embodiment of the present disclosure relates to a method for evaluating the therapeutic and / or preventive effect of a substance or agent on a skin disease, the skin disease being a skin disease caused by obstruction and / or inflammation of the pilosebaceous system. Hereinafter, this embodiment will also be referred to as the evaluation method of the first embodiment. In one aspect, the evaluation method of the first embodiment includes, in this order, contacting a substance or agent with sebaceous gland cells or their precursor cells (contacting step) and evaluating the sebaceous gland cells or their precursor cells (evaluating step).

[0019] The substance or agent to be evaluated in the evaluation method of the first embodiment is not particularly limited and may be a substance or agent that has or may have a therapeutic and / or preventive effect on a skin disease. For example, the substance to be evaluated or a substance contained in the agent to be evaluated may be a substance that can be or may be contained in a medicine as an ingredient in a topical preparation or other preparation, and the substance may be a medicinal ingredient. The substance to be evaluated or a substance contained in the agent to be evaluated may be a substance with unknown medicinal effect. The substance or agent to be evaluated may be one type, or a mixture of two or more types.

[0020] In the contacting step, a substance or agent is contacted with sebaceous gland cells or their precursor cells. Here, the sebaceous gland cells or their precursor cells contacted with the substance or agent in the contacting step may be, for example, cells present in an animal or isolated cells. Below, the contacting step will be described separately for the case where the sebaceous gland cells or their precursor cells are cells present in an animal and the case where the sebaceous gland cells are isolated cells.

[0021] In one embodiment, the sebocytes or their precursor cells contacted with the substance or agent in the contacting step are cells possessed by an animal. In this case, the animal may be, for example, an animal other than a human, preferably a mammal or avian animal, more preferably a mammalian animal, and even more preferably a rodent (Rodentia). Furthermore, when the animal according to this embodiment is a mammalian animal, it may be, for example, a mouse, rat, guinea pig, hamster, ferret, gerbil, rabbit, dog, or monkey, or may be a mouse, rat, guinea pig, hamster, ferret, or gerbil, or may be a mouse, rat, guinea pig, or hamster, or may be a mouse or rat.

[0022] In this embodiment, the substance or agent is administered to an animal to contact the substance or agent with sebaceous gland cells or their precursor cells in the animal. In this case, the method of administering the substance or agent to the animal may be any method that allows the substance or agent to contact the sebaceous gland cells or their precursor cells in the animal, and may be, for example, transdermal, oral, intravenous, subcutaneous, intramuscular, intraperitoneal, nasal, or ocular administration. The substance or agent may be formulated to suit the administration form. The dosage of the substance or agent may be determined depending on the substance or agent, and may be, for example, 1 pg or more and 1 kg or less per animal. The time interval between the contacting step and the subsequent step can be appropriately set and optimized by those skilled in the art depending on the substance or agent and the administration form, and may be, for example, 1 hour or more and 30 days or less.

[0023] In another embodiment, the sebaceous gland cells or their precursor cells contacted with the substance or agent in the contacting step are isolated cells. Here, the isolated cells are not limited to isolated cells consisting only of sebaceous gland cells or their precursor cells, but may also be isolated cells containing sebaceous gland cells or their precursor cells, i.e., the isolated cells may include cells other than sebaceous gland cells or their precursor cells. When the sebaceous gland cells or their precursor cells contacted with the substance or agent in the contacting step are isolated cells, the isolated cells may be cells isolated by collection from the above-mentioned animals, or may be a cultured cell line derived from the above-mentioned animals or humans, and in one embodiment, may be a cultured cell line derived from humans, or may be a cultured cell line derived from human skin cells. In a preferred embodiment, the skin cells may be cells derived from sebaceous glands or hair follicle keratinocytes.

[0024] In this embodiment, the sebaceous gland cells or their precursor cells are contacted with the substance or agent by adding the substance or agent to the culture medium for culturing the sebaceous gland cells or their precursor cells. The culture medium can be appropriately selected and optimized by those skilled in the art depending on the type and origin of the sebaceous gland cells or their precursor cells. For example, a culture medium that can be used for culturing the sebaceous gland cells or their precursor cells to proliferate may be used. The substance or agent can be added to the culture medium by adding the substance or agent to the culture medium, and the substance or agent may be appropriately formulated or modified to permeate the cell membrane. The concentration of the substance or agent in the culture medium may be determined depending on the cells and the substance or agent, and may be, for example, 1 pM to 1 M. The contact conditions in the contacting step can be appropriately set and optimized by those skilled in the art depending on the cells and the substance or agent. For example, the contact time may be 1 hour to 30 days.

[0025] In one aspect, the evaluation method of the first embodiment further comprises contacting sebaceous gland cells or their precursor cells with a C14 to C18 fatty acid and / or an alkyl ester thereof before, after, or simultaneously with the contacting step, and before the evaluation step (fatty acid contacting step). In one aspect, when the sebaceous gland cells or their precursor cells contacted with a substance or agent in the contacting step are cells present in an animal, if the evaluation method of the first embodiment comprises a fatty acid contacting step, a pilosebaceous gland-derived skin disease is developed at the site where the fatty acid is applied. In this case, the site where the C14 to C18 fatty acid and / or an alkyl ester is applied may overlap with the site where the substance or agent was contacted in the contacting step. In another aspect, when the sebaceous gland cells or their precursor cells contacted with a substance or agent in the contacting step are isolated cells, if the evaluation method of the first embodiment comprises a fatty acid contacting step, the onset process of a pilosebaceous gland-derived skin disease is reproduced in the sebaceous gland cells or their precursor cells.

[0026] The C14 to C18 fatty acid refers to a fatty acid having 14 or more and 18 or less carbon atoms. In a preferred embodiment, the C14 to C18 fatty acid may be a C16 fatty acid. When the fatty acid is a C16 fatty acid, inflammation can be induced highly efficiently in sebocytes or their precursor cells.

[0027] The C14 to C18 fatty acids in the present disclosure may be saturated or unsaturated fatty acids, and if they are unsaturated fatty acids, the position and stereochemistry of the carbon-carbon double bonds are not limited. Furthermore, the number of carbon-carbon double bonds contained in the C14 to C18 fatty acids in the present disclosure may be, for example, 0 to 3, 0 to 2, or 0 to 1. In a preferred embodiment, the number of carbon-carbon double bonds contained in the C14 to C18 fatty acids in the present disclosure may be 0, i.e., the C14 to C18 fatty acids may be saturated fatty acids. When the C14 to C18 fatty acids are saturated fatty acids, inflammation can be induced with high efficiency in pilosebaceous skin diseases. Furthermore, the C14 to C18 fatty acids in the present disclosure may be straight-chain fatty acids. In a preferred embodiment, the C14 to C18 fatty acids do not contain any carbon-carbon triple bonds.

[0028] Examples of C14 to C18 fatty acids in the present disclosure include myristic acid (14:0), pentadecanoic acid (15:0), palmitic acid (16:0), heptadecanoic acid (17:0), stearic acid (18:0), myristoleic acid (14:1), sapienic acid (16:1), palmitoleic acid (16:1), oleic acid (18:1), elaidic acid (18:1), vaccenic acid (18:1), petroselinic acid (18:1), isostearic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), pinolenic acid (18:3), eleostearic acid (18:3), calendic acid (18:3), punicic acid (18:3), and stearidonic acid (18:4). The C14 to C18 fatty acid according to one embodiment may be one or more fatty acids selected from the group consisting of myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid, stearic acid, sapienic acid, isostearic acid, and oleic acid, may be one or more fatty acids selected from the group consisting of myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid, and stearic acid, may be myristic acid, palmitic acid, or stearic acid, or may be palmitic acid.

[0029] The alkyl ester of a C14 to C18 fatty acid in the present disclosure may be, for example, a C1 to C3 alkyl ester of a C14 to C18 fatty acid. When the alkyl ester of a C14 to C18 fatty acid in the present disclosure is a C1 to C3 alkyl ester of a C14 to C18 fatty acid, in one embodiment the C1 to C3 alkyl ester is a methyl ester, ethyl ester, n-propyl ester, or isopropyl ester. In a preferred embodiment, the C1 to C3 alkyl ester is an isopropyl ester or ethyl ester, and in a more preferred embodiment, the C1 to C3 alkyl ester is an ethyl ester. Furthermore, the alkyl ester of a C14 to C18 fatty acid in the present disclosure may be a straight-chain alkyl ester of a straight-chain fatty acid.

[0030] In a preferred embodiment of the present disclosure, the C14 to C18 fatty acid and / or alkyl ester thereof is one or more selected from the group consisting of myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid, stearic acid, isostearic acid, methyl myristate, methyl pentadecanoate, methyl palmitate, methyl heptadecanoate, methyl stearate, methyl isostearate, ethyl myristate, ethyl pentadecanoate, ethyl palmitate, ethyl heptadecanoate, ethyl stearate, ethyl isostearate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, and isopropyl isostearate. In a more preferred embodiment of the present disclosure, the C14 to C18 fatty acid and / or alkyl ester thereof is one or more selected from the group consisting of ethyl myristate, ethyl pentadecanoate, ethyl palmitate, ethyl heptadecanoate, ethyl stearate, ethyl isostearate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, and isopropyl isostearate. In another more preferred embodiment of the present disclosure, the C14 to C18 fatty acid and / or alkyl ester thereof is one or more selected from the group consisting of palmitic acid, isopropyl palmitate, and ethyl palmitate. In a further preferred embodiment of the present disclosure, the C14 to C18 fatty acid is palmitic acid, and the alkyl ester is an ethyl ester. In an even more preferred embodiment of the present disclosure, the C14 to C18 fatty acid and / or alkyl ester thereof is ethyl palmitate.

[0031] In one embodiment, when the sebaceous gland cells or their precursor cells contacted with the substance or agent in the contacting step are cells present in an animal, the C14 to C18 fatty acid and / or its alkyl ester according to the present disclosure may be applied to the animal's skin in a state dissolved, suspended, or emulsified in a solvent. The solvent is not particularly limited, and examples thereof include water, ethanol, isopropyl alcohol, glycerin, butylene glycol, propylene glycol, polyethylene glycol, acetone, methanol, dimethylformamide, pyridine, dimethyl sulfoxide, and mixtures thereof. In one embodiment, the solvent may be ethanol, a mixture of water and ethanol, acetone, a mixture of acetone and water, a mixture of water and propylene glycol, a mixture of water and polyethylene glycol, or a mixture of water and dimethyl sulfoxide.

[0032] In one embodiment, when the sebocytes or their precursor cells contacted with the substance or agent in the contacting step are cells present in an animal, the amount of C14 to C18 fatty acid and / or alkyl ester thereof applied to the animal's skin per application may be, for example, 0.0750 to 3.60 mg, 0.250 to 1.80 mg, or 0.450 to 1.13 mg, based on the mass of the C14 to C18 fatty acid and / or alkyl ester. Furthermore, the absolute amount of C14 to C18 fatty acid and / or alkyl ester thereof applied to the animal's skin per application may be, for example, 0.250 to 13.0 μmol, 0.750 to 6.50 μmol, or 1.50 to 4.00 μmol, based on the C14 to C18 fatty acid and / or alkyl ester. Furthermore, when the C14 to C18 fatty acid and / or its alkyl ester is applied in a state of being dissolved, suspended or emulsified in a solvent, the amount of the solvent to be applied may be, for example, 5 to 50 μL, 10 to 30 μL, or 15 to 25 μL.

[0033] In another aspect, when the sebocytes or their precursor cells contacted with the substance or agent in the contacting step are isolated cells, the fatty acid contacting step can be carried out in the same manner as described in the contacting step, except that C14 to C18 fatty acids and / or their alkyl esters are contacted instead of the substance or agent.

[0034] The evaluation method of one aspect of the first embodiment includes a fatty acid contacting step before or simultaneously with the contacting step. In this case, the therapeutic effect of the substance or agent to be evaluated on pilosebaceous gland-derived skin diseases is suitably evaluated. Furthermore, the evaluation method of another aspect of the first embodiment includes a fatty acid contacting step after or simultaneously with the contacting step. In this case, the preventive effect of the substance or agent to be evaluated on pilosebaceous gland-derived skin diseases is suitably evaluated.

[0035] The evaluation step includes a step of evaluating sebaceous gland cells or their precursor cells after the contacting step or after the contacting step and the fatty acid contacting step. Specifically, for example, the evaluation step first involves preparing a sample for evaluation from sebaceous gland cells or their precursor cells after the contacting step or after the contacting step and the fatty acid contacting step (sample preparation operation), and then evaluating the prepared sample (sample evaluation operation). Such a sample is, for example, a solution containing the biomolecule to be evaluated, or sebaceous gland cells or their precursor cells.

[0036] In one aspect, when the sebaceous gland cells or their precursor cells that are contacted with a substance or agent in the contacting step are cells that an animal has, the sample preparation operation involves collecting tissue containing sebaceous gland cells or their precursor cells from the animal after the contacting step or after the contacting step and the fatty acid contacting step, and preparing a sample from that tissue.

[0037] The collection of tissue containing sebaceous gland cells or their precursor cells may be performed by, for example, excising and recovering tissue containing sebaceous gland cells or their precursor cells from the skin of an animal. The collected tissue may be a tissue containing a site contacted with a substance or agent. Furthermore, when the evaluation method according to one aspect of the first embodiment is a fatty acid contacting step, the collected tissue may be a tissue containing a site contacted with a C14 to C18 fatty acid and / or an alkyl ester thereof.

[0038] The method for preparing a sample from tissue can be selected appropriately by those skilled in the art depending on the evaluation target and evaluation method in the subsequent sample evaluation operation. For example, when evaluating the amount of a biomolecule in a population of sebaceous gland cells or their precursor cells contained in tissue in the sample evaluation operation, the sample can be prepared from the tissue by first lysing the tissue and then extracting the gene, protein, or lipid to be evaluated from the lysate, and these can be performed according to methods commonly used by those skilled in the art. For example, when evaluating the amount of a biomolecule in each cell of sebaceous gland cells or their precursor cells contained in tissue in the sample evaluation operation, the sample can be prepared from tissue according to methods commonly used in single-cell RNA-seq analysis, for example, according to the method described in Non-Patent Document 3. In this case, the cells used as the sample may include cells other than sebaceous gland cells or their precursor cells, or may consist of sebaceous gland cells or their precursor cells.

[0039] In another aspect, when the sebocytes or their precursor cells contacted with a substance or agent in the contacting step are isolated cells, and, for example, when evaluating the amount of a biomolecule in a population of sebocytes or their precursor cells contained in the cells, the sample preparation procedure can be performed in the same manner as described above for preparing a sample from tissue collected from an animal, except that cells are used instead of tissue. On the other hand, when the sebocytes or their precursor cells contacted with a substance or agent in the contacting step are isolated cells, and, for example, when evaluating the amount of a biomolecule in each of the sebocytes or their precursor cells contained in the cells, the evaluation step may not include a sample preparation procedure. In this case, the sebocytes or their precursor cells themselves serve as the sample in the subsequent sample evaluation procedure.

[0040] In the sample evaluation operation, sebaceous gland cells or their precursor cells are evaluated by evaluating the sample. The subject of evaluation in the sample evaluation operation may be appropriately selected by those skilled in the art depending on the purpose of the evaluation. In one aspect, the sample evaluation operation may evaluate the amount of at least one element selected from the group consisting of genes, proteins, and lipids in sebaceous gland cells or their precursor cells. In other words, in one aspect, the evaluation step may include evaluating the amount of at least one element selected from the group consisting of genes, proteins, and lipids in sebaceous gland cells or their precursor cells. In another aspect, the evaluation step may include evaluating at least one element selected from the group consisting of genes and proteins in sebaceous gland cells or their precursor cells, or may include evaluating at least one gene in sebaceous gland cells or their precursor cells. The evaluation of genes, proteins, and lipids may, for example, evaluate the presence and / or amount of genes, proteins, and lipids. The evaluation of genes, proteins, and lipids may be performed according to a method appropriately selected or optimized by those skilled in the art depending on the subject of evaluation. For example, gene evaluation may be performed using a lysate sample by measurement using a next-generation sequencer, measurement using a microarray, or measurement by reverse transcription PCR, or may be performed according to a method commonly used in single-cell RNA-seq analysis, such as the method described in Non-Patent Document 3. Protein evaluation may also be performed using a lysate sample by ELISA or Western blotting, or the target protein in a cell sample may be fluorescently labeled by fusion with a fluorescent protein or immunofluorescent staining, followed by fluorescence imaging or flow cytometry. For example, lipid evaluation may be performed by mass spectrometry or chromatography, and in one embodiment, by LC-MS.In addition, when evaluating cells one by one in the evaluation process, if the cells include cells other than sebaceous gland cells or their precursor cells, for example, only cells that are positive for one or more genes (e.g., Lrig1) or proteins (e.g., Lrig1 protein) that are typically expressed by sebaceous gland cells or their precursor cells may be evaluated as sebaceous gland cells or their precursor cells.

[0041] In one embodiment, the sample evaluation procedure may assess the amount of a marker associated with keratinization and / or inflammation in sebocytes or their precursor cells. In a preferred embodiment, the sample evaluation procedure may assess the amount of a keratinization marker and / or a chemokine-related marker in sebocytes or their precursor cells. In other words, in a preferred embodiment, the evaluation step may include assessing the amount of a keratinization marker and / or a chemokine-related marker in sebocytes or their precursor cells. In addition, when a specific gene name is described as a marker whose amount is to be evaluated in the present disclosure, the marker refers to mRNA.

[0042] A cornification marker is a gene whose expression level is significantly increased or decreased in keratinized epidermal keratinocytes compared to non-keratinized epidermal keratinocytes, and in one embodiment, is such an mRNA. The keratinization marker according to the present disclosure may be any gene known to exhibit a significant difference in expression level depending on the presence or absence of keratinization, and may be, for example, Krt17 (Keratin 17) and / or Krt16 (Keratin 16). From the viewpoint of being easily usable as a marker due to its high degree of expression increase in sebocytes or their precursor cells, a keratinization marker according to a preferred embodiment of the present disclosure may include or be Krt17.

[0043] A chemokine-related marker is a chemokine that promotes an inflammatory response, a biological molecule that assists in such promotion, or a gene for such a chemokine; in one embodiment, it is a chemokine that promotes an inflammatory response or its gene; in a preferred embodiment, it is the gene for a chemokine that promotes an inflammatory response; and in a more preferred embodiment, it is the mRNA of a chemokine that promotes an inflammatory response. The chemokine-related marker according to the present disclosure may be any marker known to exhibit a significant difference in expression level depending on whether or not an inflammatory response is promoted by a chemokine, and may be, for example, at least one selected from the group consisting of CXCL1 (C-X-C motif chemokine ligand 1), CXCL2 (C-C motif chemokine ligand 2), CCL2 (C-C motif chemokine ligand 2), CCL7 (C-C motif chemokine ligand 7), IL-1A (Interleukin-1α), IL-1B (Interleukin-1β), IL-6 (Interleukin-6), IL-17A (Interleukin-17A), IL-17F (Interleukin-17F), IFNA (Interferon-α), IFNG (Interferon-γ), and TNF (Tumor Necrosis Factor). From the viewpoint that the expression level increases greatly in sebocytes or their precursor cells and that they can be easily used as a marker, a chemokine-related marker according to a preferred embodiment of the present disclosure may include or may be CXCL1.

[0044] When the evaluation method according to one embodiment does not include a fatty acid contact step, the evaluation method according to this embodiment may evaluate, for example, the amount of a marker related to keratinization and / or inflammation in sebocytes or their precursor cells, and a substance or agent that shows results showing inhibition of keratinization and / or inflammation or a reduced probability of their occurrence compared to when the contact step is not included may be evaluated as having a therapeutic and / or preventive effect on pilosebaceous skin diseases.

[0045] When the evaluation method according to one embodiment includes a fatty acid contacting step before or simultaneously with the contacting step, the evaluation method according to this embodiment may evaluate, for example, the amount of a marker associated with keratinization and / or inflammation in sebocytes or their precursor cells, and evaluate a substance or agent that gives results showing suppression of keratinization and / or inflammation compared to when the contacting step is not included, as having a therapeutic effect on pilosebaceous skin diseases. Furthermore, when the evaluation method according to one embodiment includes a fatty acid contacting step after or simultaneously with the contacting step, the evaluation method according to this embodiment may evaluate, for example, the amount of a marker associated with keratinization and / or inflammation in sebocytes or their precursor cells, and evaluate a substance or agent that gives results showing suppression of keratinization and / or inflammation compared to when the contacting step is not included, as having a preventive effect on pilosebaceous skin diseases.

[0046] A second embodiment of the present disclosure relates to a method for screening for a substance or agent that prevents and / or treats a skin disease. Hereinafter, this embodiment will also be referred to as the "screening method of the second embodiment." The screening method of the second embodiment includes a contacting step and an evaluation step. The contacting step of the second embodiment may be performed in the same manner as the evaluation method of the first embodiment, except that a candidate substance or candidate agent is contacted instead of a substance or agent. Here, the candidate substance or candidate agent may be the same as the substance or agent related to the evaluation method of the first embodiment. The evaluation step of the second embodiment may also be performed in the same manner as the evaluation method of the first embodiment. The screening method of the second embodiment according to one aspect may further include a fatty acid contacting step. In this case, the fatty acid contacting step may be performed in the same manner as the evaluation method of the first embodiment. The order of the steps and the resulting evaluation aspects are also the same as those of the evaluation method of the first embodiment.

[0047] The screening method of the second embodiment may include, after the evaluation step, a step (selection step) of selecting a substance or agent that prevents and / or treats a pilosebaceous gland-derived skin disease based on the evaluation results of the evaluation step. In one aspect, in the selection step, a substance or agent in which the amount of a marker associated with keratinization and / or inflammation in sebocytes or their precursor cells evaluated in the evaluation step has changed in a direction indicating inhibition of keratinization and / or inflammation compared to a case in which the contact step has not been included may be selected as a substance or agent that prevents and / or treats a pilosebaceous gland-derived skin disease, and in a preferred aspect, a substance or agent in which the change is significant or noticeable may be selected as a substance or agent that prevents and / or treats a pilosebaceous gland-derived skin disease.

[0048] A third embodiment of the present disclosure relates to a method for discovering targets for the treatment and / or prevention of skin diseases. Hereinafter, this embodiment will also be referred to as the "discovery method of the third embodiment." The discovery method of the third embodiment includes artificially increasing, decreasing, or inhibiting the amount or activity of a protein (hereinafter also referred to as a "candidate target protein") that is a candidate target for the treatment and / or prevention of pilosebaceous gland-derived skin diseases, with sebocytes or their precursor cells as target cells (perturbation step) in cells or animals containing sebocytes or their precursor cells. Furthermore, the discovery method of the third embodiment according to one aspect may include, after the perturbation step, a step of evaluating cells containing sebocytes or their precursor cells in which the amount or activity of the candidate target protein has been artificially increased, decreased, or inhibited (cell evaluation step). The discovery method according to this aspect may further include a fatty acid contacting step before the cell evaluation step. In this case, the fatty acid contacting step may be performed in the same manner as the evaluation method of the first embodiment.

[0049] The present inventors have found that in the early stages of the onset of pilosebaceous skin diseases, keratinization and / or inflammatory signaling are particularly enhanced in sebocytes or their precursor cells. Therefore, if the protein that induces the enhancement of keratinization and / or inflammatory signaling in such sebocytes or their precursor cells can be identified, it is believed that treatment and / or prevention can be achieved by directly approaching the mechanism of onset of pilosebaceous skin diseases using a drug that targets that protein.

[0050] The candidate target protein may be any protein expressed in sebaceous gland cells or their precursor cells. In one embodiment, the candidate target protein may be a protein that is relatively highly expressed in sebaceous gland cells or their precursor cells among cells that form skin tissue, a protein that is relatively highly expressed in sebaceous gland cells or their precursor cells among cells that form hair follicle tissue, or a protein that is selectively expressed in sebaceous gland cells or their precursor cells among cells that form hair follicle tissue. In a preferred embodiment, the candidate target protein may be a protein whose expression level increases in sebaceous gland cells or their precursor cells at the early stage of a pilosebaceous gland-derived skin disease, or a protein that is or may be involved in inflammation in sebaceous gland cells or their precursor cells. The protein according to such a preferred embodiment may be a protein described in a publicly known publication (e.g., Non-Patent Document 2) that reports the protein or gene expression profile of sebaceous gland cells or their precursor cells.

[0051] In a preferred embodiment, the candidate target protein may be a cell membrane receptor. When the candidate target protein is a cell membrane receptor, a candidate target protein can be found that can target the stage in which a ligand binds to the cell membrane receptor, which occurs at the earliest stage in the onset of pilosebaceous gland-derived skin diseases. The cell membrane receptor may be any receptor expressed in sebocytes or their precursor cells, and may be, for example, a biological membrane receptor such as a G protein-coupled receptor, a fatty acid-binding protein, a fatty acid transport protein, a scavenger receptor, or a free fatty acid receptor, or a nuclear receptor.

[0052] In the perturbation step, artificially increasing, decreasing, or inhibiting the amount or activity of a candidate target protein in cells or animals containing sebocytes or their precursor cells can be performed according to techniques commonly used by those skilled in the art, and these can be performed on the whole body or part of an animal containing sebocytes or their precursor cells, or on isolated sebocytes or their precursor cells. The amount of the candidate target protein can be increased, for example, by genetically overexpressing the protein in the whole body or part of an animal containing sebocytes or their precursor cells, or in isolated sebocytes or their precursor cells. The amount of the protein can be reduced, for example, by knockdown, knockout, or contact with an antibody specific to the protein. The activity of the protein can be increased, for example, by adding an enhancer or by genetic modification that improves the activity of the protein. The activity of the protein can be reduced, for example, by genetic modification that reduces the activity of the protein. The activity of the protein can be inhibited, for example, by adding or administering a small molecule inhibitor.

[0053] The cell evaluation step includes a step of evaluating cells containing sebaceous gland cells or their precursor cells after the perturbation step, or after the perturbation step and the fatty acid contact step. The "cells containing sebaceous gland cells or their precursor cells" evaluated in the cell evaluation step may be sebaceous gland cells or their precursor cells, or may be a population of cells containing sebaceous gland cells or their precursor cells and other cells. The cells to be evaluated may include a portion of cells in which the amount or activity of a target candidate protein has been artificially increased, decreased, or inhibited in the perturbation step, or may include all of the cells. When the cells to be evaluated are sebaceous gland cells or their precursor cells, the evaluation in the cell evaluation step can be performed in the same manner as the evaluation step described in the first embodiment. When the cells to be evaluated are cells that are a population containing sebocytes or their precursor cells and other cells, the evaluation may be carried out according to a method commonly used in evaluating skin cells, such as gene expression analysis by measurement using a next-generation sequencer after lysing, measurement using a microarray, or measurement by reverse transcription PCR, protein expression analysis by ELISA or Western blotting after lysing, protein expression analysis by fluorescent imaging or flow cytometry, or lipid profile analysis by mass spectrometry or chromatography after lysing.

[0054] In the screening method of the third embodiment, the amount or activity of a candidate target protein in sebocytes or their precursor cells is artificially increased, decreased, or inhibited in the perturbation step. As a result, in the screening method according to one aspect of the third embodiment, candidates in which the amount of a marker related to keratinization and / or inflammation in cells containing sebocytes or their precursor cells has changed in a direction indicating worsening or suppression of keratinization and / or inflammation, compared to when cells containing sebocytes or their precursor cells that have not been artificially increased, decreased, or inhibited are evaluated, can be found as proteins that can be targets (sites of action) for the treatment and / or prevention of pilosebaceous skin diseases. [Example]

[0055] The present disclosure will be described in more detail below using examples, but the present disclosure should not be construed as being limited to the following examples.

[0056] [Reference Example 1: Creation and evaluation of skin disease model mice] We investigated whether application of ethyl palmitate to mouse ears would induce skin diseases. Specifically, we investigated whether palmitic acid secretion is a cause of acne vulgaris, and whether exposure of the skin to palmitic acid would cause or exacerbate acne vulgaris.

[0057] Ethyl palmitate (Tokyo Chemical Industry Co., Ltd.) in 1, 3, 5, or 10 wt% ethanol was applied to the ears of C57BL / 6J mice at 20 μL / day per ear once daily for a total of 5 days, starting on day 0. As a negative control, ethanol was applied to the ears of C57BL / 6J mice at 20 μL / day per ear for a total of 5 days, starting on day 0. On day 5, tissues from the treated area were harvested and fixed in 10% neutral buffered formalin (Fujifilm Wako Pure Chemical Industries, Ltd.) in PBS and embedded in paraffin. The fixed tissues were cut into 4 μm-thick sections and stained with hematoxylin and eosin. Images were taken using an all-in-one fluorescence microscope BZ-X710 (Keyence, Osaka, Japan). The results are shown in Figure 1. The cross-sectional area of ​​hair follicles formed in mice treated with ethanol and mice treated with a 5 or 10 wt% ethanol solution of ethyl palmitate was measured perpendicular to the epidermis at 15 locations per mouse for three mice. The results are shown in Figure 2. The results in Figure 2 are shown as mean ± standard deviation (n = 3). In the figure, * indicates a P value of less than 0.05 in one-way analysis of variance, and *** indicates a P value of less than 0.001 in one-way analysis of variance.

[0058] Subsequently, total RNA was isolated from hair follicles formed in mice treated with ethanol and mice treated with 1, 5, or 10 wt% ethanolic ethyl palmitate solutions using TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA) and the Direct-zol RNA Miniprep Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's instructions, including the area where ethyl palmitate was applied. RNA concentration was assessed using a Cytation™ 3 microplate reader (BioTek, Winooski, VT, USA). RNA extracted from mouse ears was reverse-transcribed using a High Capacity cDNA kit (Applied Biosystems, Foster City, CA, USA). Gene quantification was performed by quantitative real-time PCR. The primers used are listed in Table 1. qPCR was performed using a ViiA7 real-time PCR system (Life Technologies Corporation, Grand Island, NY, USA). Gene abundance was calculated using the dCT method. Gene abundance was normalized to the abundance of Gapdh mRNA, a housekeeping gene. Results for the inflammatory marker Il-1α are shown in Figure 3(a). Results for the inflammatory marker Il-1β are shown in Figure 3(b). Results for the inflammatory marker Tnf are shown in Figure 3(c). Results for the inflammatory marker Defb4 are shown in Figure 3(d). Results for the inflammatory marker Defb14 are shown in Figure 4(a). Results for the inflammatory marker S100a8 are shown in Figure 4(b). Results for the inflammatory marker S100a9 are shown in Figure 4(c). Results for the inflammatory marker Tslp are shown in Figure 4(d). Results for the hyperkeratosis marker Krt16 are shown in Figure 5(a). Results for the hyperkeratosis marker Klk6 are shown in Figure 5(b). Results for the hyperkeratosis marker Krt17 are shown in Figure 5(c). These results are shown as mean ± standard error (n = 3).These results were also tested using one-way analysis of variance, and in the figures, * indicates a P value of less than 0.05, ** indicates a P value of less than 0.01, *** indicates a P value of less than 0.001, and **** indicates a P value of less than 0.0001.

[0059] [Table 1]

[0060] As shown in Figure 1, clear comedo formation was observed in the groups to which 3, 5, and 10 wt% ethyl palmitate ethanol solutions were applied. Furthermore, as shown in Figure 2, the size of hair follicles in mice to which 5 and 10 wt% ethyl palmitate ethanol solutions were applied was significantly larger than that in mice to which ethanol was applied. These results strongly suggest that application of 5 and 10 wt% ethyl palmitate ethanol solutions resulted in comedo formation and enlargement of hair follicles. As shown in Figures 3 and 4, an increase in all inflammatory markers was observed in the groups to which 5 and 10 wt% ethyl palmitate ethanol solutions were applied. Furthermore, as shown in Figure 5, an increase in hyperkeratosis markers, which indicate the occurrence of hyperkeratosis, a precursor to skin diseases caused by hyperkeratinization, was observed in the groups to which 5 and 10 wt% ethyl palmitate ethanol solutions were applied. This strongly suggests that application of ethyl palmitate promotes comedo formation by inducing hyperkeratinization. The above results demonstrate that the model mice prepared in this Reference Example are suitable as models for skin diseases caused by obstruction and / or inflammation of the pilosebaceous system.

[0061] Example 1: Single-cell RNA-seq analysis of early-stage skin disease model mice Mice were prepared by application of a 5 wt% ethanol solution of ethyl palmitate, as in Reference Example 1. Cells were isolated from tissues containing the ethyl palmitate-applied area of ​​mice (controls) that were not treated with ethyl palmitate, and from mice treated with the ethanol solution 6 and 12 hours after application. Tissue processing was performed according to the method published in Non-Patent Document 3. For single-cell RNA-seq, cells were pooled from five mice per group and sorted. Prior to sorting, cells were labeled with the following antibodies: CD45-FITC (Biolegend), EpiCAM-APC (Biolegend), and eBioscience™ Fixable Viability Dye eFluor™ 780 (Invitrogen). Sorting was performed using a CellSorter SH800ZFP (SONY). The isolated cells were pooled by group at a ratio of 5:2.5 (keratinocytes; CD45-EpiCAM+):2.5 (immune cells; CD45+):2.5 (others; CD45-EpiCAM-). Single-cell RNA-seq libraries were constructed using the Chromium Next GEM Single Cell 3' Library and Gel Bead Kit v3.1 (10x Genomics) according to the manufacturer's instructions. Libraries were sequenced using NovaSeq X plus (Illumina) with a read length of 28x90, yielding a minimum of 20,000 reads per cell. The resulting raw data were processed using Cell Ranger 7.1.0 (10x Genomics). Further analysis was performed using the Seurat R package (v4.2.1) (Butler, A. et al., “Integrating single-cell transcriptomic data across different conditions, technologies, and species.”, Nature Biotechnology 36(5), 411-420(2018).) with default parameters unless otherwise specified.For quality control purposes, analysis was limited to cells with a mitochondrial gene ratio of less than 5% and a total number of expressed genes ranging from 200 to 5,000. Data were normalized using the NormalizeData function, and variably expressed genes were identified using the FindVariableFeatures function. Principal components (PCs) were calculated from these variably expressed genes using the RunPCA function after scaling with the default parameters of the ScaleData function. Clusters were identified using the FindNeighbors and FindClusters functions with a resolution of 0.1. Nonlinear dimensionality reduction and visualization were performed using UMAP with the RunUMAP function. Based on marker genes, clusters were classified into 14 cell types: keratinocytes, T cells, fibroblast-1, fibroblast-2, myeloid cells, hair follicle keratinocytes, dendritic cell-1, dendritic cell-2, innate lymphoid cells, endothelial cells, sebocytes, mast cells, smooth muscle, and other. Further sub-clustering was performed using the FindSubCluster function with an argument of resolution=0.1. "Hair follicle keratinocytes" were further sub-clustered into 4 clusters, and "sebaceous gland cells" were further sub-clustered into 3 clusters. In this way, the final Seurat object was created.

[0062] Figure 6 shows the results of single-cell RNA-seq analysis, plotting the expression profile after reducing its dimensionality using the Uniform Manifold Approximation and Projection (UMAP). Figure 7 shows the expression levels of various markers used to identify the origin of cells in mouse skin tissue in the cells plotted in Figure 6. Figure 8 shows the expression levels of various markers used to identify the origin of cells in mouse skin tissue in the cells plotted in Figure 6, visualized for each classified cluster. Based on the results of Figures 7 and 8, the origin of cells was assigned to each cluster in Figure 6. Figure 9 shows the plots for the control, 6 hours after application, and 12 hours after application, which are the basis for Figure 6. As shown in Figures 6 to 9, single-cell RNA-seq analysis roughly divided the cells into 14 clusters.

[0063] Figure 10 shows the expression levels of various markers in four cell populations (4_0-4_3) further classified within the cluster classified as hair follicle keratinocyte-derived cells and three cell populations (10_0-10_2) further classified within the cluster classified as sebaceous gland-derived cells, as determined by single-cell RNA-seq analysis. As shown in Figure 10, one Lrig1-positive cell population (4_1) was observed in the cluster classified as hair follicle keratinocyte-derived cells and one in the cluster classified as sebaceous gland-derived cells (10_1). These two cell populations were located near the sebaceous gland infundibulum, suggesting that they primarily contain sebocytes and their progenitor cells.

[0064] Figure 11 compares the expression levels of various markers in the two Lrig1-positive cell populations identified in Figure 10 with those in epidermal keratinocytes. As shown in Figure 11, 6 and 12 hours after application of ethyl palmitate, the expression levels of Krt17, a keratinization marker of hair follicle keratinocytes, and Cxcl1, an inflammatory chemokine-related marker, were elevated in the two Lrig1-positive cell populations. In contrast, no increase in the expression levels of these keratinization markers or inflammatory chemokine-related markers was observed in clusters other than sebaceous glands and hair follicle keratinocytes, including epidermal keratinocytes. These results demonstrate that in the early stages of pilosebaceous gland-derived skin diseases, inflammation occurs in sebaceous glands and hair follicle keratinocytes located near the sebaceous infundibulum. Because sebaceous glands are lipid-secreting tissues, it was highly unexpected that the lipids they secrete could actually cause inflammation in the sebaceous glands.

Claims

1. A method for evaluating the therapeutic and / or preventive effect of a substance or agent on a skin disease, comprising: contacting a substance or agent with sebocytes or their precursor cells; and evaluating the sebocytes or their progenitor cells; The method, wherein the skin disease is a skin disease resulting from blockage and / or inflammation of the pilosebaceous system.

2. A method for screening a substance or agent that prevents and / or treats a skin disease, comprising: contacting a candidate substance or agent with a sebocyte or a precursor cell thereof; and evaluating the sebocytes or their progenitor cells; The method, wherein the skin disease is a skin disease resulting from blockage and / or inflammation of the pilosebaceous system.

3. A method for discovering targets for treating and / or preventing skin diseases, comprising: artificially increasing, decreasing or inhibiting the amount or activity of a protein that is a candidate target for the treatment and / or prevention of the skin disease, in cells or animals having sebocytes or their precursor cells, with sebocytes or their precursor cells as target cells; The method, wherein the skin disease is a skin disease resulting from blockage and / or inflammation of the pilosebaceous system.

4. The method according to any one of claims 1 to 3, wherein the sebocytes or precursor cells thereof are cells that are present in an animal.

5. The method according to any one of claims 1 to 3, wherein the sebocytes or their precursor cells are isolated cells.

6. The method according to any one of claims 1 to 3, wherein the sebocytes or precursor cells thereof are sebaceous precursor cells.

7. The method according to any one of claims 1 to 3, wherein the sebocytes or their precursor cells are Lrig1-positive cells.

8. The method according to any one of claims 1 to 3, wherein the skin disease is an inflammatory skin disease resulting from hyperkeratosis.

9. The method according to any one of claims 1 to 3, wherein the skin disease is acne vulgaris.

10. The method according to any one of claims 1 to 3, wherein evaluating the sebocytes or their precursor cells comprises evaluating the amount of at least one selected from the group consisting of genes, proteins, and lipids in the sebocytes or their precursor cells.

11. The method according to any one of claims 1 to 3, wherein evaluating the sebocytes or their precursor cells comprises evaluating the amount of a keratinization marker and / or a chemokine-related marker in the sebocytes or their precursor cells.

12. The method of claim 11 , wherein the keratinization marker comprises Krt17.

13. The method of claim 11 , wherein the chemokine-associated marker comprises Cxcl1.

14. The method according to any one of claims 1 to 3, further comprising contacting the sebocytes or their precursor cells with a C14 to C18 fatty acid and / or an alkyl ester thereof.

15. 15. The method of claim 14, wherein the C14 to C18 fatty acid is palmitic acid.

16. 15. The method of claim 14, wherein the alkyl ester is an ethyl ester.

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