Modeling method and application of psoriasis-like skin organoid

Abstract

The invention relates to a modeling method and application of psoriasis-like skin organs, and belongs to the technical field of medical experiments. The invention provides a modeling method of psoriasis-like skin organs, which comprises the following steps: adding pancreatin into the back skin of a mouse for enzymolysis to obtain enzymolysis skin, separating dermis and epidermis of the enzymolysis skin, respectively digesting by collagenase to obtain dermis cells and epidermis cells, and carrying out enzymolysis on the dermis cells and the epidermis cells to obtain the psoriasis-like skin organs. Mixing the dermal cells and the epidermal cells, culturing to obtain pre-cultured cells, continuously culturing the pre-cultured cells by adopting an inflammatory factor culture medium, and detecting indexes to screen the psoriasis-like skin organs. The psoriasis-like skin organoid constructed by the invention not only can be used for researching the effect of immune cells in psoriasis progress, but also can reveal the roles of other skin cells in a disease process, so that a more comprehensive model is provided for in-vitro research of psoriasis, deep understanding of pathogenesis of psoriasis is facilitated, and the development of psoriasis is promoted. And a foundation is laid for developing a new treatment strategy.

Description

Technical Field

[0001] The present invention relates to the field of medical experimental technology, and in particular to a modeling method and application of psoriasis-like skin organoids. Background Art

[0002] Psoriasis is a chronic, relapsing, inflammatory, systemic immune-mediated disease induced by both genetic and environmental factors. Genetic factors are the primary risk factor for psoriasis, while cell-related immune pathways such as interleukin-23 (IL-23) and T helper 17 (Th17) cells are the core mechanisms of psoriasis pathogenesis. Dendritic cells are considered the primary driver of psoriasis. Activated dendritic cells can produce IL-12 and IL-23, inducing the activation or proliferation of Th1, Th17, and Th22 cells, producing cytokines such as TNF-α, IL-17, and IL-22. This stimulates excessive proliferation of keratinocytes and the production of related cytokines and chemokines, thereby establishing an inflammatory cycle.

[0003] Organoids are three-dimensional cell aggregates derived from stem cells or organ progenitor cells that can differentiate and self-organize to form specific functions and structures that have some of the corresponding organs in the human body. Because organoids are humanized, they can simulate organ development and generation, have genomic stability during long-term expansion in vitro, and can form a living biobank for high-throughput screening, making them an in vitro model that has attracted much attention. Using organoid models to mimic the 3D tissue structure and physiological cell behavior of healthy and disease models can provide valuable information for the establishment of disease models at the organ level. However, the current cell models for simulating psoriasis are still immature. Keratinocytes are a common 2D cell model in psoriasis research. They have poor reproducibility, availability and stability, making it difficult to fully understand the pathogenesis of psoriasis. 3D cell models are an extension of 2D cell models that can better simulate the three-dimensional structure of tissues and the physiological and pathological changes after the interaction of multiple cells, but they lack the overall organ structure and are insufficient to simulate the physiological functions at the organ level.

[0004] Currently, commonly used methods for constructing in vitro models of psoriasis include treating keratinocytes with inflammatory factors, co-culturing immune cells and keratinocytes, and culturing psoriatic epidermal and dermal cells. Because the pathogenesis of psoriasis involves the interaction of multiple major cell types, the interaction between a single cell type and keratinocytes is insufficient to reveal the core mechanisms of psoriasis progression. Existing technologies urgently need to develop a more complex three-dimensional in vitro model that can accurately simulate the pathophysiological processes of psoriasis by simulating the interactions between different cell types in the skin. Summary of the Invention

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for modeling psoriasis-like skin organoids, comprising the following steps: (1) The skin of the mouse back was taken and enzymatically hydrolyzed with trypsin for 12-14 hours to obtain enzymatically hydrolyzed skin; (2) Separate the dermis and epidermis of the enzymatically hydrolyzed skin by digesting them with collagenase for 15 to 45 minutes, filtering and centrifuging to obtain dermal cells and epidermal cells; (3) Mixing dermal cells and epidermal cells and inoculating them into culture wells, adding culture medium and culturing for 2 to 4 days to obtain pre-cultured cells; (4) The pre-cultured cells were inoculated into the culture wells, and inflammatory factor culture medium was added to continue culturing for 3 to 6 days. The indicators were tested to screen for psoriasis-like skin organoids.

[0006] Preferably, the concentration of the pancreatic enzyme in step (1) is 0.1% to 0.5%, and the amount of pancreatic enzyme added is 10 to 30 mL; The temperature of the enzymatic hydrolysis is 1-5°C.

[0007] Preferably, the digestion temperature in step (2) is 35-40°C; The centrifugal temperature is 2-6° C., the centrifugal time is 3-10 min, and the centrifugal speed is 1000-1500 rpm.

[0008] Preferably, the ratio of the mixed dermal cells and epidermal cells in step (3) is 5 to 10:1; The inoculation amount is 3 to 6×10 7 cells / well; The culture medium is DMEM / F12 culture medium, and the amount of the culture medium added is 500-1000 μL / well; The culture temperature is 35-40°C.

[0009] Preferably, the inflammatory factor culture medium in step (4) uses water as a solvent and includes the following components at the following concentrations: IL-17 10-15 μmol / L, IL-23 10-15 μmol / L, TNF-α 5-10 μmol / L, IL-1A 5-10 μmol / L, and Galectin 92-5 μg / mL.

[0010] Preferably, the amount of the inflammatory factor culture medium added in step (4) is 700-800 μL / well; The temperature for continued cultivation is 35-40°C.

[0011] The present invention also provides application of the modeling method in preparing psoriasis-like skin organoids.

[0012] The present invention has the following technical effects and advantages: The present invention adds Galectin-9 and inflammatory factors such as IL-17, IL-23, TNF-α to the 3D culture technology, thereby constructing a psoriasis-like skin organoid that is closer to the clinical pathological characteristics. Among them, 3D culture technology can better simulate the real skin environment, including the three-dimensional arrangement of cells, cell-cell and cell-matrix interactions; using inflammatory factors such as IL-17, IL-23, TNF-α as key stimulating factors in the process of constructing psoriasis-like skin organoids can simulate the inflammatory response process of psoriasis in vitro; since dendritic cells are considered to be the main driving factor for the onset of psoriasis, the addition of Galectin-9 can accurately simulate the function and role of dendritic cells in psoriasis. The number and ratio of cells used in the present invention, as well as the culture medium formula composed of different inflammatory factors are the result of multiple attempts, optimization and testing. Psoriasis-like skin organoids cannot be constructed outside the concentration range of the present invention; The psoriasis-like skin organoids of the present invention contain major cell types in the skin, such as dermal fibroblasts, epidermal cells, dendritic cells, and T cells. By stimulating the occurrence of psoriasis through inflammatory factor culture medium, it can solve the problem of lack of research models for the promotion of pathological processes by interactions between multiple cells during the onset of psoriasis. It can not only be used to study the role of immune cells in the progression of psoriasis, but also reveal the role of other skin cells in the disease process, thereby providing a more comprehensive model for in vitro research on psoriasis, helping to deeply understand the pathogenesis of psoriasis and laying the foundation for the development of new treatment strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 A process for constructing psoriasis-like skin organoids; Figure 2 Timeline for culture medium replacement in psoriatic skin organoids; Figure 3 The results of single cell RNA-sequencing analysis of psoriasis-like skin organoids are shown; Figure 4 This is the immunofluorescence staining identification result of psoriasis-like skin organoids; Figure 5 Results of epidermal thickness measurements of psoriatic skin organoids; Figure 6 The results show the expression levels of inflammatory factors in psoriasis-like skin organoids. DETAILED DESCRIPTION

[0014] The present invention provides a method for modeling psoriasis-like skin organoids, comprising the following steps: (1) Taking the skin from the back of a mouse, adding trypsin and hydrolyzing it for 12 to 14 hours, preferably 13 hours, to obtain enzymatically hydrolyzed skin; (2) separating the dermis and epidermis of the enzymatically hydrolyzed skin, digesting them with collagenase for 15 to 45 minutes, preferably 30 minutes, filtering and centrifuging to obtain dermal cells and epidermal cells; (3) mixing the dermal cells and epidermal cells and inoculating them into culture wells, adding culture medium and culturing for 2 to 4 days, preferably 3 days, to obtain pre-cultured cells; (4) The pre-cultured cells are inoculated into the culture wells, and inflammatory factor culture medium is added and cultured for 3 to 6 days, preferably 4 days, and psoriasis-like skin organoids are screened by detecting indicators.

[0015] In the present invention, the concentration of the pancreatic enzyme in step (1) is 0.1% to 0.5%, preferably 0.25%; the amount of pancreatic enzyme added is 10 to 30 mL, preferably 20 mL; The temperature of the enzymatic hydrolysis is 1-5°C, preferably 4°C.

[0016] In the present invention, the digestion temperature in step (2) is 35-40°C, preferably 37°C; The centrifugal temperature is 2-6°C, preferably 4°C; the centrifugal time is 3-10 min, preferably 5 min; the centrifugal speed is 1000-1500 rpm, preferably 1100 rpm.

[0017] In the present invention, the ratio of the mixed dermal cells and epidermal cells in step (3) is 5 to 10:1, preferably 9:1; The inoculation amount is 3 to 6×10 7 cells / well, preferably 5×10 7 cells / well; The culture medium is DMEM / F12 culture medium, and the amount of the culture medium added is 500-1000 μL / well, preferably 700 μL / well; The culture temperature is 35-40°C, preferably 37°C.

[0018] In the present invention, the inflammatory factor culture medium described in step (4) uses water as a solvent and includes the following components at the following concentrations: IL-17 10-15 μmol / L, preferably 13 μmol / L; IL-23 10-15 μmol / L, preferably 12 μmol / L; TNF-α 5-10 μmol / L, preferably 8 μmol / L; IL-1A 5-10 μmol / L, preferably 8 μmol / L; Galectin9 2-5 μg / mL, preferably 3 μg / L.

[0019] In the present invention, the amount of the inflammatory factor culture medium added in step (4) is 700-800 μL / well, preferably 750 μL / well; The temperature for continued cultivation is 35-40°C, preferably 37°C.

[0020] In the present invention, the indicators described in step (4) include epidermal thickness and inflammatory factor expression levels.

[0021] The present invention also provides application of the modeling method in preparing psoriasis-like skin organoids.

[0022] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0023] Among the test animals of the present invention, ICR mice weighed 25-30 g and were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.; Among the reagents of the present invention, trypsin (catalog number: T4549) was purchased from Sigma, USA; collagenase (catalog number: LS004197) was purchased from Worthington, USA; and DMEM / F12 culture medium (catalog number: 10092018) was purchased from Corning, USA. In the instrument of the present invention, the transwell chamber culture system (Article No.: TCS016012) was purchased from JET Company, China.

[0024] Example 1: Construction of psoriasis-like skin organoids (1) The dorsal skin of ICR mice was cut with ophthalmic scissors, and the connective tissue was gently scraped off with curved forceps to obtain the dorsal skin. The skin was then spread flat on a 10 cm culture dish and 20 mL of 0.25% EDTA-free trypsin was added. The skin was enzymatically hydrolyzed at 4°C for 13 h to obtain the enzymatically hydrolyzed skin. (2) Using pointed forceps, the dermis and epidermis of the enzymatically hydrolyzed skin were separated and digested with collagenase at 37°C for 30 min. After filtration, the cells were centrifuged at 1100 rpm for 5 min to obtain dermal cells and epidermal cells. (3) Dermal cells and epidermal cells were mixed at a ratio of 9:1 and inoculated into the culture wells of a 12-well transwell chamber culture system. The inoculation volume was 5×10 7 10 cells / well, add 700 μL DMEM / F12 medium to the bottom of each inoculation well of the transwell chamber, culture at 37°C for 3 days to obtain pre-cultured cells, and replace the DMEM / F12 medium every other day during the culture period; (4) 13 μmol IL-17, 12 μmol IL-23, 8 μmol TNF-α, 8 μmol IL-1A, and 3 μg Galectin9 were added to 1 L of water to obtain an inflammatory factor culture medium; (5) The pre-cultured cells were inoculated into the culture wells, 750 μL of inflammatory factor culture medium was added, and the cells were cultured at 37°C for 4 days. The inflammatory factor culture medium was replaced every other day during the culture period. (6) Single cell RNA-sequencing analysis and immunofluorescence staining were used to identify the tissue structure, and indicators such as epidermal thickness and inflammatory factor expression were detected to screen psoriasis-like skin organoids; Figures 1-2 shown.

[0025] Experimental Example 1: Single Cell RNA-Sequencing Analysis Shenzhen BGI Genomics Co., Ltd. was commissioned to perform single cell RNA-sequencing analysis on the psoriasis-like skin organoids constructed in Example 1. The results are as follows: Figure 3 shown.

[0026] The results showed that the psoriasis-like skin organoids constructed by the present invention contained dermal fibroblasts, epidermal cells, endothelial cells, melanocytes, neurons, adipocytes, myofibroblasts, dendritic cells, T cells, and mast cells.

[0027] Experimental Example 2: Immunofluorescence staining identification Immunofluorescence staining was performed on the psoriasis-like skin organoids constructed in Example 1, and the results were as follows: Figure 4 shown.

[0028] The results showed that dermal cells (VIM), epidermal cells (K14), epidermal cells (ColXVII), endothelial cells (CD31), and monocytes (F13A1) could be identified from the psoriasis-like skin organoids constructed by the present invention.

[0029] Experimental Example 3: Epidermal Thickness Measurement The psoriasis-like skin organoids constructed in Example 1 were subjected to H&E staining, and the epidermal thickness of the psoriasis-like skin organoids was measured using imageJ software. Normal skin was used as a control group. The results are as follows: Figure 5 shown.

[0030] The results showed that the epidermal thickness of the psoriasis-like skin organoids constructed by the present invention was significantly increased compared with the control group.

[0031] Experimental Example 4: Determination of inflammatory factor expression The gene expression levels of psoriasis-related inflammatory factors such as Tnf, IL-23, IL-17a, IL-22, and IL-1b in the psoriasis-like skin organoids constructed in Example 1 were measured by RT-qPCR, and normal skin was used as the control group. The results are as follows Figure 6 shown.

[0032] The results showed that the expression levels of various inflammatory factors in the psoriasis-like skin organoids constructed by the present invention were significantly upregulated compared with the control group ( p <0.05).

[0033] As can be seen from the above examples, the present invention provides a method for modeling and application of psoriasis-like skin organoids. The psoriasis-like skin organoids of the present invention contain dermal fibroblasts, epidermal cells, dendritic cells, T cells and other major cell types in the skin. By stimulating the occurrence of psoriasis through inflammatory factor culture medium, they can address the problem of the lack of research models for the interaction between multiple cells in the pathogenesis of psoriasis and the promotion of pathological progression. They can not only be used to study the role of immune cells in the progression of psoriasis, but also reveal the role of other skin cells in the disease process, thereby providing a more comprehensive model for in vitro research on psoriasis, contributing to a deeper understanding of the pathogenesis of psoriasis and laying the foundation for the development of new treatment strategies.

[0034] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for modeling psoriasis-like skin organoids, characterized in that: The steps include: (1) The skin of the mouse back was taken and enzymatically hydrolyzed with trypsin for 12-14 hours to obtain enzymatically hydrolyzed skin; (2) Separate the dermis and epidermis of the enzymatically hydrolyzed skin by digesting them with collagenase for 15 to 45 minutes, filtering and centrifuging to obtain dermal cells and epidermal cells; (3) Mixing dermal cells and epidermal cells and inoculating them into culture wells, adding culture medium and culturing for 2 to 4 days to obtain pre-cultured cells; (4) The pre-cultured cells were inoculated into the culture wells, and inflammatory factor culture medium was added to continue culturing for 3 to 6 days. The indicators were tested to screen for psoriasis-like skin organoids.

2. The modeling method according to claim 1, wherein The concentration of the pancreatic enzyme in step (1) is 0.1% to 0.5%, and the amount of pancreatic enzyme added is 10 to 30 mL; The temperature of the enzymatic hydrolysis is 1-5°C.

3. The modeling method according to claim 2, wherein: The digestion temperature in step (2) is 35-40°C; The centrifugal temperature is 2-6° C., the centrifugal time is 3-10 min, and the centrifugal speed is 1000-1500 rpm.

4. The modeling method according to claim 3, wherein: The ratio of the mixed dermal cells and epidermal cells in step (3) is 5 to 10:1; The inoculation amount is 3 to 6×10 7 cells / well; The culture medium is DMEM / F12 culture medium, and the amount of the culture medium added is 500-1000 μL / well; The culture temperature is 35-40°C.

5. The modeling method according to claim 4, characterized in that: The inflammatory factor culture medium described in step (4) uses water as a solvent and includes the following components at the following concentrations: IL-17 10-15 μmol / L, IL-23 10-15 μmol / L, TNF-α 5-10 μmol / L, IL-1A 5-10 μmol / L, and Galectin9 2-5 μg / mL.

6. The modeling method according to claim 5, characterized in that: The amount of the inflammatory factor culture medium added in step (4) is 700-800 μL / well; The temperature for continued cultivation is 35-40°C.

7. Use of the modeling method according to any one of claims 1 to 6 in preparing psoriasis-like skin organoids.

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