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CN-122003604-A - Skin injury organoid model and method for screening medicines by using same

CN122003604ACN 122003604 ACN122003604 ACN 122003604ACN-122003604-A

Abstract

The present invention relates to a skin injury organoid model and a method for screening medicine using the same, which uses a technology for preparing a skin organoid by using an Air-liquid interface (Air-liquid interface), so that skin cells, accessory structures, nerve cells, fat cells, etc. form a structure similar to that of actual skin, and ultraviolet rays which simulate the actual arrival at the surface of the skin are irradiated to the skin organoid, thereby preparing the injured skin. In the present invention, the skin barrier is damaged by irradiating solar simulated ultraviolet rays including 94.5% ultraviolet rays (UV-A) and 5.5% ultraviolet rays (UV-B), and the activity of matrix proteolytic enzymes is enhanced to promote collagen decomposition while increasing the secretion of pro-inflammatory cytokines, thereby favorably simulating the skin damage characteristics caused by ultraviolet rays. Therefore, the invention can be used as a skin injury and photoaging model caused by ultraviolet rays and can be applied to the screening of potential therapeutic drugs.

Inventors

  • KANG JINGXUAN
  • JIN MINZHI

Assignees

  • 康干细胞生物科技有限公司

Dates

Publication Date
20260508
Application Date
20241008
Priority Date
20231010

Claims (20)

  1. 1. A method for preparing a skin lesion organoid comprising the steps of: (1) Culturing a multipotent stem cell-derived organoid in the presence of a Wnt agonist; (2) Culturing the culture of step (1) in a skin organoid maturation medium; (3) Cutting the culture of step (2) and culturing at gas-liquid interface to prepare skin organoids, and (4) Irradiating the skin organoids with ultraviolet rays simulating sunlight, Wherein the Wnt agonist is added upon induction differentiation of non-neuroectodermal layers into cranial neural crest-like cells (Cranial neural CREST CELL; CNCC).
  2. 2. The method of claim 1, wherein, The skin injury organoid is a hair follicle injury organoid with damaged hair follicles in an accessory organ of skin.
  3. 3. The method of claim 1, wherein, The Wnt agonist is selected from the group consisting of CHIR-99021, wnt3A, WNT a, and RSPO 1.
  4. 4. The method of claim 1, wherein, The Wnt agonist was added on days 5-7 of pluripotent stem cell culture.
  5. 5. The method of claim 1, wherein, And (3) cutting the culture in the step (2) into 4 uniform sizes, and performing gas-liquid interface culture on the transmembrane culture insert of the collagen coating to enable the dermis layer to face the collagen side, and exposing the epidermis layer to air.
  6. 6. The method of claim 1, wherein the step of determining the position of the substrate comprises, The skin injury organoids damage skin through NF-kB activation.
  7. 7. A skin-damaging organoid prepared by the method of claim 1.
  8. 8. A method of screening a skin damage therapeutic agent, prophylactic agent or ameliorating agent, comprising the step of treating a skin damage organoid according to claim 7 with a skin damage therapeutic agent, prophylactic agent or ameliorating agent candidate.
  9. 9. The method of claim 2, wherein the step of determining the position of the substrate comprises, The follicle damaging organoids damage the follicle through NF-kB activation.
  10. 10. The method of claim 2, wherein the step of determining the position of the substrate comprises, The hair follicle damaging organoids have reduced expression of one or more genes selected from the group consisting of dermal sheath marker α -SMA, outer root sheath marker KRT5 or KRT15, hair follicle stem cell marker KRT15 or LHX2, and hair papilla marker SOX 2.
  11. 11. The method of claim 2, wherein the step of determining the position of the substrate comprises, In the damaged hair follicle organoids, inflammation induces an increase in gene expression of the cytokines COX-2, TNF- α or IL-1β.
  12. 12. A hair follicle damaging organoid prepared by the method of claim 2.
  13. 13. A method for screening a hair growth stimulant, a hair loss preventive, a hair loss mitigant or a hair loss therapeutic agent, comprising the step of treating the hair follicle damaging organoid according to claim 12 with a hair growth stimulant, a hair loss preventive, a hair loss mitigant or a hair loss therapeutic agent candidate substance.
  14. 14. The method of claim 13, wherein the step of determining the position of the probe is performed, The candidate substance inhibits activated NF-kB in the damaged organoids of hair follicles.
  15. 15. A biomarker composition for detecting hair follicle damage, comprising NF-kB as an active ingredient.
  16. 16. A kit for detecting hair follicle damage comprising a formulation capable of detecting NF-kB.
  17. 17. The kit of claim 16, wherein, The preparation is selected from more than one of the group consisting of NF-kB specific primers, probes, antisense oligonucleotides, aptamers and antibodies.
  18. 18. A method of providing information required for hair follicle damage detection, comprising the steps of: (1) Measuring the expression level of NF-kB from the isolated sample; (2) Comparing the expression level of NF-kB with a control sample, and (3) If the expression level of NF-kB is higher than that of the control group sample, the hair follicle is judged to be damaged.
  19. 19. A pharmaceutical composition for a hair growth promoter, a hair loss preventive, a hair loss relieving agent or a hair loss therapeutic agent, which comprises an NF-kB activity or expression inhibitor as an active ingredient.
  20. 20. The pharmaceutical composition of claim 19, wherein, The NF-kB activity inhibitor is selected from any one of the group consisting of a small molecule compound, a peptide, a peptidomimetic, an aptamer, an antibody, and a natural substance that specifically binds to an NF-kB protein.

Description

Skin injury organoid model and method for screening medicines by using same Technical Field The present application claims priority from korean patent application No. 10-2023-0134562 filed 10/2023, the entire contents of which are incorporated herein by reference. The present invention relates to a skin injury organoid model and a method for screening drugs using the same, and more particularly, to a method for preparing a photoaging-like skin model caused by ultraviolet injury by irradiating differentiated human skin organoids with ultraviolet rays simulating actual arrival at the surface of the skin, and a method for evaluating the efficacy of potential therapeutic drugs. The present invention was completed by the topic number 00215812 (1711195569) with the support of the korean scientific and technical information communication unit. Background With the improvement of living standard, modern people keep healthy and pay more attention to keep healthy skin. Accordingly, there is increasing interest in not only skin beauty but also improvement in skin damage and aging. The skin is the largest organ among organs constituting the human body, accounting for about 15% of the body weight, and is an important organ which covers the body surface and is in direct contact with the outside. Skin cells are damaged by a variety of external factors that attempt to invade the body. Among them, exposure to ultraviolet rays is recognized as an exogenous aging factor of skin, and various damages can be caused to the skin by only exposing to ultraviolet rays. In recent years, the amount of ultraviolet rays reaching the earth's surface tends to increase due to the decrease in the ozone layer that plays a protective role in the uppermost part of the atmosphere, and such an increase may potentially cause skin damage. Currently, methods for improving damage and aging-related phenomena caused by ultraviolet rays are being actively studied, and particularly, studies are continuously conducted for exploring a main mechanism causing skin damage or aging and determining substances capable of alleviating the same. Prior art literature Patent literature Korean laid-open patent No. 10-2023-015654 (2023.08.03 publication). Disclosure of Invention Technical problem to be solved by the invention The present invention aims to provide a method for preparing a skin injury organoid or a hair follicle injury organoid by irradiating ultraviolet rays simulating sunlight. Furthermore, it is another object of the present invention to provide a skin injury organoid or hair follicle injury organoid prepared by the preparation method. Still another object of the present invention is to provide a method for screening a therapeutic, prophylactic or ameliorating agent for skin injury using the skin injury organoid. Still another object of the present invention is to provide a method for screening a hair growth promoter, a hair loss preventive, a hair loss mitigant or a hair loss therapeutic agent using the damaged hair follicle organoid. It is still another object of the present invention to provide a biomarker composition for detecting hair follicle damage, which comprises NF-kB as an active ingredient. Still another object of the present invention is to provide a kit for detecting hair follicle damage, which comprises a preparation capable of detecting NF-kB. Furthermore, it is another object of the present invention to provide a method for providing information required for hair follicle damage detection by measuring the expression level of NF-kB. Further, still another object of the present invention is to provide a pharmaceutical composition for a hair growth promoter, a hair loss preventive, a hair loss relieving agent or a hair loss therapeutic agent, which comprises an NF-kB activity or expression inhibitor as an active ingredient. Furthermore, it is still another object of the present invention to provide a pharmaceutical composition or cosmetic composition for depilatory, which comprises NF-kB activity or expression enhancer as an active ingredient. Furthermore, it is another object of the present invention to provide a hair growth promoting, hair loss preventing, hair loss alleviating or hair loss treating method comprising the step of administering an inhibitor of NF-kB activity or expression to an individual in need of such treatment in an effective amount. Furthermore, it is a further object of the present invention to provide a method of dehairing comprising the step of administering an NF-kB activity or expression enhancer in an effective amount to an individual in need of such treatment. Means for solving the problems In order to achieve the above object, the present invention provides a method for preparing a skin injury organoid, comprising the steps of (1) culturing a pluripotent stem cell-derived organoid in the presence of a Wnt agonist; (2) Culturing the culture of step (1) in a skin organoid maturation medium; (3) Cutting the cult