Diabetes skin lesion organoid model and construction method thereof

Abstract

The invention provides a diabetic skin lesion organoid model and a construction method and application thereof. The diabetic skin lesion organoid model is obtained by culturing a skin organoid obtained by differentiating mouse embryonic stem cells in a high-glucose environment. According to the invention, the skin organ injury model caused by high glucose is obtained by culturing the skin organ model in the high glucose environment for the first time. Researches find that the model shows typical characteristics of diabetic skin lesions such as AGEs accumulation, abnormal glycolipid metabolism, neural network damage and the like. Therefore, the model can be used for research on a diabetic skin lesion mechanism and drug screening research.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a diabetic skin lesion organoid model, a construction method thereof and an application thereof. Background Art

[0002] As an emerging biological model, organoids have shown great potential in the fields of biomedical research, drug screening, etc. The stratified skin organoids constructed by Lee et al. through the differentiation of induced pluripotent stem cells can present an almost complete stratified skin structure, and include appendages such as hair follicles and neural networks, and are ideal tools for in vitro modeling (Lee, J. et al. Nature https: / / doi.org / 10.1038 / s41586-020-2352-3 (2020)). However, there has been no report on an organoid model of diabetic skin lesions with high biomimetic degree in the prior art. Summary of the Invention

[0003] To achieve the object of the present invention, in a first aspect, the present invention provides a diabetic skin lesion organoid model, and the construction method of the organoid model comprises the following steps:

[0004] Differentiating mouse embryonic stem cells into skin organoids;

[0005] Placing the skin organoids in a high-glucose maturation medium for high-glucose culture to obtain the diabetic skin lesion organoid model; wherein, the high-glucose maturation medium comprises glucose with a final concentration of 25-75 mM; preferably, the high-glucose maturation medium comprises glucose with a final concentration of 50 mM.

[0006] In a second aspect, the present invention also provides a kit for constructing a diabetic skin lesion organoid model.

[0007] In a third aspect, the present invention also provides a construction method of a diabetic skin lesion organoid model.

[0008] In a fourth aspect, the present invention also provides an application of the diabetic skin lesion organoid model or the kit in the research on the mechanism of diabetic skin lesions and drug screening. The beneficial effect of the present invention is that the diabetic skin lesion organoid model of the present invention has typical characteristics of diabetic skin lesions such as AGEs accumulation, neural network damage and abnormal glycolipid metabolism, and has a high biomimetic degree. Description of the Drawings

[0009] Figure 1 : It is a flow chart for the present invention to study the preparation of a diabetic skin lesion organoid model;

[0010] Figure 2: Detection results of AGEs content in the diabetic skin lesion organoid model constructed in the present invention before and after injury

[0011] Figure 3 : Expression of the nerve marker TUJ1 in the diabetic skin lesion organoid model constructed in the present invention before and after injury; a. TUJ1 immunofluorescence staining map, b. Relative fluorescence area quantitative analysis;

[0012] Figure 4 : Analysis of gene expression differences in the diabetic skin lesion organoid model constructed in the present invention before and after injury; where Organoid is a normal skin organoid, and Organoid+HG (High Glucose) is a skin organoid model under high glucose treatment; a. PCA, b. Volcano plot, c. Number of differentially expressed genes;

[0013] Figure 5 : GO enrichment analysis map of up-regulated differentially expressed genes in the diabetic skin lesion organoid model constructed in the present invention before and after injury;

[0014] Figure 6 : KEGG enrichment analysis map of up-regulated differentially expressed genes in the diabetic skin lesion organoid model constructed in the present invention before and after injury;

[0015] Figure 7 : Enrichment chord diagram of glycolipid metabolism-related pathways in the diabetic skin lesion organoid model constructed in the present invention before and after injury;

[0016] Figure 8 : Screening potential therapeutic targets for diabetic skin lesions by protein-protein interaction network analysis using the diabetic skin lesion organoid model constructed in the present invention. Detailed implementation manners

[0017] Diabetic skin lesions are one of the most common and easily overlooked complications in the diagnosis and treatment of diabetes. In different regions of the world, the prevalence of skin lesions in type 1 and type 2 diabetes patients ranges from 51.1% to 97%. As the disease progresses, diabetic skin lesions can lead to severe complications such as peripheral nerve dysfunction, skin ulceration and infection, and chronic ulcers, causing great harm to the physical and mental health of patients. In-depth exploration of the occurrence and development mechanism of diabetic skin lesions is of great significance for early identification and diagnosis of diabetes and avoiding the occurrence of severe complications.

[0018] Studies have shown that the main pathophysiological characteristics of diabetic skin lesions are abnormal skin structure, chronic inflammation, and microenvironmental imbalance caused by glucose metabolism disorders. Pathological hyperglycemic state inhibits keratinocyte proliferation and migration, reduces protein biosynthesis, induces endothelial cell apoptosis, reduces nitric oxide production, and impairs the phagocytosis and chemotaxis of several functional cells. These changes affect skin homeostasis and lead to the loss of normal biological functions. In addition, the production and accumulation of advanced glycation end products (AGEs) caused by hyperglycemia are another major factor in the occurrence of diabetic skin lesions. AGEs are formed by the glycation of proteins, lipids, and nucleic acids. It can induce the formation of reactive oxygen species (ROS) and damage protein functions inside and outside cells. In addition to changing the properties of collagen, reducing its flexibility and solubility, and increasing its rigidity, AGEs are also involved in diabetes-related immunosuppression, affecting skin structure and function through imbalances in growth factor secretion and activity disorders. The nerve damage caused by the hyperglycemic environment is also worthy of attention and can be used as a screening indicator for early diabetic damage. Sensory neuropathy reduces the sensitivity of the skin to the external environment, greatly increasing the incidence of diabetic ulcers.

[0019] At present, the exploration of diabetic skin lesions has developed from ordinary 2D cultured cell models to diabetic animal models, among which db / db mice and streptozotocin-induced diabetic mouse models are the most commonly used. However, animal models are often costly, time-consuming to model, difficult to control the onset time and severity, and have limited detection methods. How to establish an accurate and controllable in vitro skin model has become the key to solving the problem. With the continuous development of biotechnology, organoids, as a new type of biological model, have shown great potential in biomedical research, drug screening and other fields. The stratified skin organoids constructed by Lee et al. through the differentiation of induced pluripotent stem cells can present an almost complete stratified skin structure and include appendages such as hair follicles and neural networks, which are ideal tools for in vitro modeling. In this context, the present invention designs a method for constructing an organoid model of diabetes-related skin lesions induced by high glucose. This organoid model of diabetic skin lesions has typical characteristics of diabetic skin lesions such as AGEs accumulation, neural network damage, and abnormal glycolipid metabolism, with a high degree of biomimesis. It solves the problems of the lack of in vitro models of diabetic skin lesions, low modeling efficiency of animal models, poor model uniformity, and cumbersome modeling process.

[0020] Sources of reagents, culture media, kits, instruments, or consumables, etc.:

[0021] R1 mouse embryonic stem cells (mESCs) (Cyagen Biosciences, MUAES-01001); TrypLE cell digestive enzyme (TrypLE TMExpress Enzyme(1X), no phenol red, Thermo Fisher); Matrigel (Matrigel - 20 degrees, 2446 cell culture differentiation, German Biofroxx biological reagent); Bone Morphogenetic Protein 4 (BMP - 4) (PeproTech); SB - 431542 is a TGF - β receptor kinase inhibitor (TRKI), with the molecular formula C 22 H 16 N4O3 (Stemgent); Low - dose naltrexone (LDN) (Stemgent); Fibroblast Growth Factor - 2 (FGF - 2) (PeproTech); Mouse Advanced Glycation End - products (AGEs) ELISA Kit (Wuhan Saipai Biotechnology Co., Ltd., SP14643); Mouse β - tubulin III (TUJ1) antibody, BioLegend, 801202); TRIzol (Thermo Fisher, 15596018); Micro - nucleic acid and protein quantifier NanoDrop ND - 1000 (NanoDrop, Wilmington, DE, USA); Agilent Bioanalyzer 2100 bio - analyzer (Agilent, CA, USA); oligo(dT) magnetic beads (Dynabeads TM Oligo(dT), cat.25 - 61005, ThermoFisher, USA); Magnesium ion fragmentation kit( Magnesium RNA Fragmentation Module, cat.E6150S, USA); Reverse transcriptase (Invitrogen TM SuperScript II Reverse Transcriptase, cat.1896649, CA, USA); E.coli DNA polymerase I (NEB, cat.m0209, USA); RNase H (Ribonuclease H) (NEB, cat.m0297, USA); dUTP Solution (dUTP solution) (Thermo Fisher, cat.R0133, CA, USA); UDG enzyme (Uracil - DNA glycosylase) (NEB, cat.m0280, MA, US); illumina Novaseq TM 6000 gene sequencer (Hangzhou Lianchuan Biotechnology Co., Ltd.);

[0022] DMEM / F12 (Gibco); Neurobasal Medium (Gibco); B-27 Supplement (Gibco); GlutaMAX Supplement (Gibco); Leukemia Inhibitory Factor (PeproTech); PD0325901 (Stemgent); CHIR99021 (Stemgent); GMEM Medium (Gibco); Knockout TM Serum Replacement (Gibco); MEM Non-Essential Amino Acid Solution 11140050 (Gibco); Sodium Pyruvate (Gibco); 2-Mercaptoethanol (Gibco); Antimicrobial Reagent (Invivogen); Advanced DMEM / F12 Medium (Gibco); N2 Supplement (Gibco); GlutaMAX TM Supplement (Gibco);

[0023] 6-well Transparent Plate (6-well Cell Culture Plate) (Nunclon Sphera); 96-well Low Cell Adhesion U-bottom Plate (Nunclon Sphera, Thermo Scientific); 24-well Low Cell Adhesion Bottom Plate (24-well Cell Culture Plate) (Nunclon Sphera).

[0024] Glossary of Terms:

[0025] Cutadapt refers to a software tool for processing various sequencing data; HISAT2 refers to a genomic alignment software tool; StringTie refers to software for RNA-Seq data analysis; RIN value (RNA integrity number) is an index developed by Agilent for evaluating the integrity of RNA, ranging from 1 to 10, where 1 represents the most severely degraded RNA and 10 represents the most intact RNA, and the RIN value is calculated when detecting total eukaryotic RNA using an Agilent 2100 Bioanalyzer; advanced glycation end products (AGEs). "1×" represents the standard concentration, that is, prepared according to the standard ratio in the instruction manual or formula. If the standard concentration of a certain supplement is 1000 ug / ml, then 1× supplement means the concentration of this supplement in the culture medium is 1000 ug / ml. "Final concentration" refers to the final concentration of the solute in the culture medium.

[0026] The present invention will be described below in conjunction with some embodiments.

[0027] In some embodiments, the organoid model of diabetic skin lesions of the present invention is obtained by culturing skin organoids differentiated from mouse embryonic stem cells in a high-glucose environment. The "high-glucose environment" includes, but is not limited to, culturing the skin organoids in a high-glucose maturation medium for high-glucose culture.

[0028] In some embodiments, in combination with Figure 1 As shown, the organoid model of diabetic skin lesions of the present invention exhibits typical characteristics of diabetic skin lesions such as AGEs accumulation, abnormal glycolipid metabolism, and neural network damage. The "typical characteristics" refer to that the accumulation content of AGEs is higher than the normal value, the key marker β3-tubulin (TUJ1) of neural network damage is higher than the normal expression level, and the expression levels of genes related to abnormal glycolipid metabolism are up-regulated or down-regulated.

[0029] In some embodiments, the method for constructing an organoid model of diabetic skin lesions includes the following steps:

[0030] Direct the differentiation of mouse embryonic stem cells into skin organoids; In some embodiments of the present invention, the source and differentiation method of the skin organoids are not limited. For example, skin organoids differentiated from induced pluripotent stem cells (iPSCs), adult stem cells (ASCs), and human embryonic stem cells (hESCs) are all within the scope of selection of the present invention.

[0031] Culture the skin organoids in a high-glucose maturation medium.

[0032] The present invention will be further described below in combination with some further embodiments.

[0033] In some further embodiments, an organoid model of diabetic skin lesions is involved, wherein the method for constructing the organoid model includes the following steps:

[0034] Differentiate mouse embryonic stem cells into skin organoids;

[0035] Culture the skin organoids in a high-glucose maturation medium for high-glucose culture to obtain the organoid model of diabetic skin lesions; wherein, the high-glucose maturation medium includes glucose with a final concentration of 25-75 mM and a maturation medium; preferably, the high-glucose maturation medium includes glucose with a final concentration of 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 mM. In these technical solutions, the maturation medium is not specifically limited, and known basal media are all within the scope of selection of the present invention.

[0036] In some further embodiments, an organoid model of diabetic skin lesions is involved, and differentiating mouse embryonic stem cells into skin organoids includes the following steps:

[0037] The passage culture of the mouse embryonic stem cells (mESCs) is carried out using the LIF-2i medium to obtain passage mouse embryonic stem cells; in these examples, the mESCs are cultured using the LIF-2i medium on a gelatin-coated transparent plate, and the LIF-2i medium is replaced every other day. The cell passage rate is maintained at 70-80%, and the mESCs of 30 generations or lower are used for subsequent experiments. In these technical solutions, the "LIF-2i medium" is not specifically limited, and known basal media are within the selection scope of the present invention. In a further technical solution, the "LIF-2i medium" is composed of DMEM / F12 and Neurobasal Medium mixed in a volume ratio of (1-5):(1-5), and simultaneously added with 0.1-1.0×N2 supplement, 0.1-1.0×B-27 supplement, 0.5-2×GlutaMAX supplement, 500-2000 U / ml leukemia inhibitory factor, 0.5-2 μM PD0325901, 1-5 μM CHIR99021, and 50-200 μg / ml Normocin. In a further technical solution, the "LIF-2i medium" is composed of DMEM / F12 and Neurobasal Medium mixed in a volume ratio of (1, 2, 3, 4 or 5):(1, 2, 3, 4 or 5), and simultaneously added with 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0×N2 supplement, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0×B-27 supplement, 0.5, 1.0, 1.5 or 2×GlutaMAX supplement, 500, 1000 or 2000 U / ml leukemia inhibitory factor, 0.5, 1.0, 1.5 or 2 μM PD0325901, 1, 2, 3, 4 or 5 μM CHIR99021, and 50, 100, 150 or 200 μg / ml Normocin.

[0038] The differentiation culture of the passage mouse embryonic stem cells is carried out using the basic ectoderm differentiation medium or the ectoderm differentiation medium to obtain cell aggregates; the ectoderm differentiation medium includes the basic ectoderm differentiation medium, and one or more of Matrigel, BMP-4, SB431542, LDN, and FGF-2; in these technical solutions, the "basic ectoderm differentiation medium (sometimes simply referred to as 'ectoderm differentiation medium')" is not specifically limited, and known basal media are within the selection scope of the present invention. In a further technical solution, the components of the "basic ectoderm differentiation medium" include: GMEM medium, and 1-3% (v / v) Knockout TM serum substitute (wherein, "v / v" represents KnockoutTM The volume ratio of serum substitute to GMEM medium (the same below), 1 - 3×MEM non-essential amino acid solution, sodium pyruvate with a final concentration of 1 - 3 mM, 2-mercaptoethanol with a final concentration of 0.05 - 0.25 mM, and Normocin with a final concentration of 50 - 200 μg / ml. In a further technical solution, the components of the "basic ectoderm differentiation medium" include: GMEM medium, and 1, 2, or 3% (v / v) Knockout TM serum substitute, 1, 2, or 3×MEM non-essential amino acid solution, sodium pyruvate with a final concentration of 1, 2, or 3 mM, 2-mercaptoethanol with a final concentration of 0.05, 0.1, 0.15, 0.20, or 0.25 mM, and Normocin with a final concentration of 50, 100, 150, or 200 μg / ml. In a further technical solution, there is also the "ectoderm differentiation medium containing Matrigel", which, on the basis of the "basic ectoderm differentiation medium", further contains 2 - 6% (v / v) Matrigel (where "v / v" represents the volume ratio of Matrigel to the "mature medium", the same below). In a further technical solution, in the "ectoderm differentiation medium containing Matrigel", it contains 2, 3, 4, 5, or 6% (v / v) Matrigel.

[0039] In a further technical solution, there is also the "ectoderm differentiation medium containing BMP-4 and 5 μM SB431542", which, on the basis of the "basic ectoderm differentiation medium", further contains 25 - 75 ng / mL of BMP-4 and 1 - 10 μM of SB431542. In a further technical solution, in the "ectoderm differentiation medium containing BMP-4 and 5 μM SB431542", it contains 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 ng / mL of BMP-4 and 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM of SB431542.

[0040] In a further technical solution, there is also the "ectoderm differentiation medium containing BMP-4 and 5 μM SB431542", which, on the basis of the "ectoderm differentiation medium containing Matrigel", further contains a final concentration of 5 - 20 ng / mL of BMP-4 and a final concentration of 0.5 - 2 μM of SB431542. In a further technical solution, in the "ectoderm differentiation medium containing BMP-4 and 5 μM SB431542", it contains a final concentration of 5, 10, 15, or 20 ng / mL of BMP-4 and a final concentration of 0.5, 1, 1.5, or 2 μM of SB431542.

[0041] In a further technical solution, it also includes an "ectoderm differentiation medium containing BMP-4 and 5 μM SB431542", which, based on the "basic ectoderm differentiation medium", further contains 2-10 μM of LDN and 50-300 ng / mL of FGF-2. In a further technical solution, in the "ectoderm differentiation medium containing BMP-4 and 5 μM SB431542", it contains 2, 3, 4, 5, 6, 7, 8, 9 or 10 μM of LDN and 50, 100, 150, 200, 250 or 300 ng / mL of FGF-2.

[0042] In a further technical solution, it also includes an "ectoderm differentiation medium containing LDN and FGF-2", which, based on the "ectoderm differentiation medium containing BMP-4 and 5 μM SB431542", further contains LDN with a final concentration of 0.5-2 μM and FGF-2 with a final concentration of 10-40 ng / mL. In a further technical solution, in the "ectoderm differentiation medium containing LDN and FGF-2", it contains LDN with a final concentration of 0.5, 1, 1.5 or 2 μM and FGF-2 with a final concentration of 10, 15, 20, 25, 30, 35 or 40 ng / mL.

[0043] The cell aggregates are matured using a maturation medium or a maturation medium containing Matrigel to obtain the skin organoids. In these technical solutions, the "maturation medium" is not specifically limited, and known basal media are all within the scope of selection of the present invention. In a further technical solution, the components of the "maturation medium" include: Advanced DMEM / F12 medium, and 1-3×N2 supplement, 1-3×GlutaMAX TM supplement and Normocin with a final concentration of 50-200 μg / ml. In a further technical solution, the components of the "maturation medium" include: Advanced DMEM / F12 medium, and 1, 2 or 3×N2 supplement, 1, 2 or 3×GlutaMAX TM supplement and Normocin with a final concentration of 50, 100, 150 or 200 μg / ml. In a further technical solution, the "maturation medium containing Matrigel" contains 2-6% (v / v) of Matrigel (where "v / v" represents the volume ratio of Matrigel to the "maturation medium", the same below). In a further technical solution, the "maturation medium containing Matrigel" contains 2, 3, 4, 5 or 6% (v / v) of Matrigel.

[0044] In some further embodiments, it relates to a diabetic skin lesion organoid model, and the high-glucose culture method includes the following steps:

[0045] First, place the skin organoids in the high - glucose maturation medium for initial culture for 1 - 5 days; then remove a part of the cultured high - glucose maturation medium and supplement it with fresh high - glucose maturation medium for supplementary culture for 6 - 18 days.

[0046] In some further embodiments, it relates to an organoid model of diabetic skin lesions, and the high - glucose maturation medium includes glucose with a final concentration of 50 mM.

[0047] In some further embodiments, it relates to an organoid model of diabetic skin lesions, and the relative content of AGEs in the organoid model of diabetic skin lesions cultured for 14 days is 1.33 times that of normal skin organoids.

[0048] In some further embodiments, it relates to an organoid model of diabetic skin lesions, and the genes related to abnormal glycolipid metabolism in the organoid model of diabetic skin lesions include APOA1, APOA2, APOA4 or APOB.

[0049] The present invention will be described below with specific embodiments.

[0050] Example 1 Construction of Skin Organoids

[0051] The "LIF - 2i medium" is composed of a 1:1 mixture of DMEM / F12 (Gibco) and Neurobasal Medium (Gibco), and at the same time, 0.5×N2 supplement (Gibco), 0.5×B - 27 supplement (Gibco), 1×GlutaMAX supplement (Gibco), 10 3 U / ml leukemia inhibitory factor (PeproTech), 1 μM PD0325901 (Stemgent), 3 μM CHIR99021 (Stemgent) and 100 μg / ml Normocin (Invivogen) are added for preparation.

[0052] The "ectoderm differentiation medium" uses GMEM medium (Gibco) and adds 1.5% (v / v) Knockout TM serum replacement (Gibco), 1×MEM non - essential amino acid solution (Gibco), 1 mM sodium pyruvate (Gibco), 0.1 mM 2 - mercaptoethanol (Gibco) and 100 μg / ml Normocin (Invivogen) for preparation.

[0053] The "maturation medium" uses Advanced DMEM / F12 medium (Gibco), and at the same time, 1×N2 supplement (Gibco), 1×GlutaMAX TMPrepared with supplements (Gibco) and 100 μg / ml Normocin (Invivogen).

[0054] Maintenance culture of mouse embryonic stem cells (mESCs):

[0055] Using a commercially purchased mouse R1 cell line, ESCs were cultured in LIF-2i medium without feeder cells. mESCs were cultured in LIF-2i medium on 6-well clear plates coated with 0.1% (w / v) gelatin. The LIF-2i medium was changed every other day, and the cell passage rate was maintained at 70 - 80%. mESCs at passage 30 or lower were used for subsequent experiments.

[0056] Inductive differentiation of skin organoids:

[0057] On day 0, mESCs were separated with 1×TrypLE Express enzyme (Gibco), resuspended in ectoderm differentiation medium, and seeded at a final concentration of 3×10 3 / 100 μL cells per well on a 96-well low-cell-adhesion U-bottom plate (Nunclon Sphera, Thermo Scientific).

[0058] On day 1, 50 μL of the ectoderm differentiation medium was removed from each well and supplemented with 50 μL of the ectoderm differentiation medium containing 4% (v / v) Matrigel (final concentration 2%, Corning).

[0059] On day 3, 25 μL of the ectoderm differentiation medium (without Matrigel) containing 50 ng / mL BMP-4 (PeproTech) and 5 μM SB431542 (Stemgent) was added to each well, making the final volume 125 μL / well. The final concentrations of BMP-4 and SB431542 were 10 ng / mL and 1 μM, respectively.

[0060] On day 4, 25 μL of the ectoderm differentiation medium (without Matrigel) containing 6 μM LDN (Stemgent) and 150 ng / mL FGF-2 (PeproTech) was added to each well, making the final volume 150 μL / well. The final concentrations of LDN and FGF-2 were 1 μM and 25 ng / mL, respectively.

[0061] On day 8, each cell aggregate was transferred to a well on a 24-well low-cell-adhesion plate (Nunclon Sphera) in 500 μL of maturation medium containing 1% (v / v) Matrigel.

[0062] Starting from the 10th day, half of the mature medium (250 μL) containing 1% (v / v) Matrigel was removed every other day and supplemented with 250 μL of mature medium without Matrigel until the 30th day to obtain skin organoids.

[0063] Example 2 Construction of an organoid model for diabetic skin lesions

[0064] On the 0th day (the 30th day of Example 1), the skin organoids were cultured in a mature medium containing 50 mM glucose.

[0065] Starting from the 2nd day (the 32nd day of Example 1), half of the mature medium (250 μL) containing 50 mM glucose was removed every other day and supplemented with 250 μL of mature medium containing 50 mM glucose until the 14th day (the 44th day of Example 1) to obtain an organoid model for diabetic skin lesions.

[0066] Control Example 1 Construction of an organoid model without diabetic skin lesions

[0067] Different from Example 2, the skin organoids were cultured in a mature medium.

[0068] Starting from the 2nd day (the 32nd day of Example 1), half of the mature medium (250 μL) was removed every other day and supplemented with 250 μL of mature medium until the 14th day (the 44th day of Example 1) to obtain an organoid model without diabetic skin lesions.

[0069] Test Example 1 Detection of AGEs content

[0070] Test method: Starting from the high-glucose treatment in Example 2, on the 0th day (the 30th day of Example 1), the 2nd day, the 4th day, the 6th day, the 8th day, the 10th day, the 12th day, and the 14th day (the 44th day of Example 1), the culture medium supernatant was collected and stored at -80°C. The determination of AGEs was performed using a mouse advanced glycation end products (AGEs) ELISA kit. The test results are as Figure 2 shown.

[0071] Compared with the control group, the relative AGEs content in the test group gradually increased, showing a significant difference starting from the 10th day. By the 14th day, the AGEs content in the test group increased by approximately 30% compared to the control group.

[0072] Test Example 2 Evaluation test for the degree of nerve injury

[0073] Test method: On the 44th day of Example 1 (the 14th day of Example 2), the skin organoids before and after high-glucose treatment were collected, embedded in paraffin, sectioned, and the expression of the nerve key marker β3-tubulin (TUJ1) was detected by immunofluorescence.

[0074] Test results:

[0075] As Figure 3 shown, on the 44th day, rich nerve distribution was visible in the control group, and the morphology of cells and nerve fibers was obvious. Compared with the control group, the expression of nerve markers in the experimental group was significantly decreased after high-glucose treatment, and the morphological changes of nerve atrophy were difficult to distinguish (a); fluorescence quantitative analysis showed that the positive area of nerve markers decreased to about 40% of the control group after high-glucose treatment (b); this proved that neuropathy occurred in this model, which was consistent with the characteristics of diabetic skin lesions.

[0076] Experiment Example 3: Test on the change of gene expression levels related to glycolipid metabolism

[0077] Experimental group: Collect the skin organoids cultured with high glucose on the 44th day of Example 1 (the 14th day of Example 2); 3 samples in each group.

[0078] Control group: Collect the skin organoids cultured in the medium without additional glucose on the 44th day of Control Example 1 (the 14th day of Control Example 1); 3 samples in each group.

[0079] Test method: Take the samples of the experimental group and the control group, and use TRIzol (thermofisher, 15596018) to isolate and purify the total sample RNA according to the operation protocol provided by the manufacturer. Then use NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA) to quality control the quantity and purity of the total RNA and detect the integrity of the RNA through Bioanalyzer 2100 (Agilent, CA, USA); a concentration > 50 ng / μL, RIN value > 7.0, and total RNA > 1 μg meet the requirements of downstream experiments. Use oligo(dT) magnetic beads (Dynabeads Oligo(dT), cat. 25-61005, ThermoFisher, USA) to specifically capture the mRNA with PolyA (polyadenylic acid) in it through two rounds of purification. Fragment the captured mRNA at high temperature using a magnesium ion fragmentation kit ( Magnesium RNAFragmentation Module, cat. E6150S, USA) for 5 - 7 minutes at 94 °C. Fragment the fragmented RNA through reverse transcriptase (Invitrogen TMcDNA was synthesized under the action of SuperScript II Reverse Transcriptase (cat.1896649, CA, USA). Then, E. coli DNA polymerase I (NEB, cat.m0209, USA) and RNase H (NEB, cat.m0297, USA) were used for second-strand synthesis to convert the double-stranded complex of these DNA and RNA into a double-stranded DNA. Meanwhile, dUTP Solution (Thermo Fisher, cat.R0133, CA, USA) was incorporated into the second strand to fill in the ends of the double-stranded DNA to blunt ends, and then an A base was added to each of its two ends to enable it to ligate with the adapter with a T base at the end. The fragment size was screened and purified using magnetic beads. The second strand was digested with UDG enzyme (NEB, cat.m0280, MA, US), and then PCR was performed - pre-denaturation at 95°C for 3 minutes, denaturation at 98°C for a total of 8 cycles, 15 seconds each, annealing to 60°C for 15 seconds, extension at 72°C for 30 seconds, and finally extension at 72°C for 5 minutes to form a library (strand-specific library) with a fragment size of 300bp ± 50bp. Finally, illumina Novaseq was used TM6000 (Hangzhou Lianchuan Biotechnology Co., Ltd.) performed paired-end sequencing on it according to the standard operation, and the sequencing mode was PE150. After using Cutadapt to filter out unqualified sequences (such as sequencing adapters and low-quality sequences) in the original data to obtain valid data (Clean Data), Hisat2 was used for alignment with the reference genome (the mouse reference genome downloaded from the NCBI official website, encoded as GRCm39, website: https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_000001635.27 / ). According to the alignment results of Hisat2, StringTie was used to reconstruct transcripts and calculate the expression levels of all genes in each sample. The gene expression level analysis mainly targeted the protein-coding genes (mRNA) annotated in the genome, counted the gene expression levels, and used this to evaluate the correlation of gene expression characteristics within and between groups of samples and the differentially expressed genes. When measuring the gene expression level, the FPKM value (Fragments Per Kilobase Million, standardized based on the original reads count of the gene) was used as the measurement index of gene expression level, and the gene expression levels in different samples were counted. Taking the fold change >= 2 (i.e., the absolute value of log2FC >= 1) and q value < 0.05 (the q value is the corrected value of the p value) as the threshold criteria for screening differentially expressed genes (|log2FC| >= 1 & q < 0.05), differentially expressed genes were obtained in the set comparison groups and enrichment analysis was performed, with a focus on changes in the expression levels of genes related to glycolipid metabolism.

[0080] Experimental results:

[0081] As Figure 4 shown, according to the results of principal component analysis (PCA), it can be seen that the gene expression patterns within the control group and the experimental group are similar, proving the reproducibility of the model, and there are obvious differences in the gene expression patterns between the groups, proving the feasibility of the model construction conditions (a); compared with the control group, there are 463 up-regulated genes and 37 down-regulated genes in the experimental group (b, c).

[0082] As Figure 5 shown, enrichment analysis was performed on the up-regulated genes in the experimental group. The GO analysis results showed that the up-regulated genes were mainly enriched in glycolipid metabolism-related entries such as lipoprotein metabolism process, cholesterol homeostasis, cholesterol efflux, low-density lipoprotein particle remodeling, and triglyceride homeostasis in biological processes.

[0083] As Figure 6As shown, enrichment analysis was performed on the up-regulated genes in the experimental group. The results of KEGG analysis showed that glycolipid metabolism-related pathways such as cholesterol metabolism, lipid and atherosclerosis, and fat digestion and absorption were significantly enriched (b). This proves that the model exhibits the characteristics of abnormal glycolipid metabolism, which is in line with the significant characteristics of diabetic skin lesions.

[0084] As Figure 7 shown, the genes involved in the above-mentioned glycolipid metabolism-related pathways were analyzed, and it was found that genes such as APOA1 / APOA2 / APOA4 / APOB were enriched in 5 or more pathways, indicating that they are key genes in the pathogenesis of diabetic skin lesions.

[0085] As Figure 8 shown, protein network interaction analysis showed that the protein encoded by the APOA1 gene plays a central role in glycolipid metabolism-related lesions. At the same time, there is an obvious interaction between PCSK9 and APOA1 / APOB. By regulating the expression of the PCSK9 gene, the expression level of APOA1 can be regulated, and PCSK9 can be used as a potential target for the prevention and treatment of diabetic skin lesions.

[0086] Based on the experimental results of Test Examples 1 to 3 above, the organoid model of diabetic skin lesions of the present invention has typical characteristics of diabetic skin lesions such as AGEs accumulation, abnormal glycolipid metabolism, and neural network damage, and has a high degree of biomimesis.

[0087] In addition, the embodiments of the present invention at least have the following advantages and beneficial effects:

[0088] (1) The organoid model of diabetic skin lesions of the present invention can adjust the added glucose concentration according to the requirements of the lesion degree, which can meet the personalized customization needs of the model. The present invention established a three-dimensional diabetic skin lesion model in vitro for the first time, which can accurately control the lesion degree and avoid the interference of other organ lesions in diabetes on the study of skin lesions. Compared with animal experiments, the cost is greatly reduced.

[0089] (2) As can be seen from Figures 5 - 8 the present invention, the organoid model of diabetic skin lesions of the present invention can be used for research such as the mechanism of diabetic skin lesions and the testing of drug effectiveness, and has broad application prospects.

[0090] (3) Key pathophysiological changes were detected to ensure the accuracy of the model, and potential therapeutic targets for diabetic skin lesions were screened out (see Figure 7 and Figure 8 ), which provides a new idea for the application of organoids in disease model construction and a powerful tool for the research and treatment of diabetic skin lesions.

[0091] The above-described embodiments are merely described as the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the spirit of the present invention's design, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A diabetic skin lesion organoid model, characterized in that, The method for constructing the organoid model comprises the following steps: Differentiating mouse embryonic stem cells into skin organoids; Placing the skin organoids in a high-glucose maturation medium for high-glucose culture to obtain the diabetic skin lesion organoid model; wherein, the high-glucose maturation medium comprises glucose with a final concentration of 25-75 mM.

2. The diabetic skin lesion organoid model according to claim 1, wherein Differentiating mouse embryonic stem cells into skin organoids comprises the following steps: Subculturing the mouse embryonic stem cells using LIF-2i medium to obtain subcultured mouse embryonic stem cells; Differentiating the subcultured mouse embryonic stem cells using a basal ectoderm differentiation medium or an ectoderm differentiation medium to obtain cell aggregates; the ectoderm differentiation medium comprises a basal ectoderm differentiation medium, and one or more of Matrigel, BMP-4, SB431542, LDN, and FGF-2; Maturing the cell aggregates using a maturation medium or a maturation medium containing Matrigel to obtain the skin organoids.

3. The diabetic skin lesion organoid model according to claim 2, wherein The high-glucose culture method comprises the following steps: First, placing the skin organoids in the high-glucose maturation medium for initial culture for 1-5 days; Then, removing a part of the cultured high-glucose maturation medium and supplementing with fresh high-glucose maturation medium for supplementary culture for 6-18 days.

4. The diabetic skin lesion organoid model according to any one of claims 1-3, wherein The LIF-2i medium is prepared by mixing DMEM / F12 and Neurobasal Medium at a volume ratio of 1-5:1-5, and simultaneously adding 0.1-1.0×N2 supplement, 0.1-1.0×B-27 supplement, 0.5-2×GlutaMAX supplement, 500-2000 U / ml leukemia inhibitory factor, 0.5-2 μM PD0325901, 1-5 μM CHIR99021, and 50-200 μg / ml Normocin; The components of the basic ectoderm differentiation medium include: GMEM medium, and 1, 2, or 3% (v / v) Knockout TM serum replacement, 1, 2, or 3×MEM non-essential amino acid solution, sodium pyruvate at a final concentration of 1, 2, or 3 mM, 2-mercaptoethanol at a final concentration of 0.05, 0.1, 0.15, 0.20, or 0.25 mM, and Normocin at a final concentration of 50, 100, 150, or 200 μg / ml; The components of the maturation medium include: Advanced DMEM / F12 medium, and 1 - 3×N2 supplement, 1 - 3×GlutaMAX supplement, and Normocin with a final concentration of 50 - 200 μg / ml. TM supplement and Normocin with a final concentration of 50 - 200 μg / ml.

5. The diabetic skin lesion organoid model according to any one of claims 1 to 3, characterized in that The high-glucose maturation medium comprises glucose with a final concentration of 50 mM.

6. The diabetic skin lesion organoid model according to any one of claims 1 to 3, characterized in that The relative content of AGEs in the diabetic skin lesion organoid model cultured for 14 days is 1.33 times that of normal skin organoids.

7. The diabetic skin lesion organoid model according to any one of claims 1 to 3, characterized in that, The genes related to abnormal glycolipid metabolism in the diabetic skin lesion organoid model include APOA1, APOA2, APOA4, or APOB.

8. A kit for constructing an organoid model of diabetic skin lesions as described in any one of claims 1 to 7, characterized in that, The kit comprises the medium for preparing the diabetic skin lesion organoid model according to any one of claims 1-6.

9. A method for constructing an organoid model of diabetic skin lesions, characterized in that, The construction method comprises the following steps: Differentiating mouse embryonic stem cells into skin organoids; Placing the skin organoids in a high-glucose maturation medium for high-glucose culture to obtain the diabetic skin lesion organoid model; wherein, the high-glucose maturation medium comprises glucose with a final concentration of 25-75 mM; preferably, the high-glucose maturation medium comprises glucose with a final concentration of 50 mM.

10. Use of the diabetic skin lesion organoid model or kit according to any one of claims 1 to 8 in the study of the mechanism of diabetic skin lesions and drug screening; preferably, the typical characteristics of diabetic skin lesions include one or more of AGEs accumulation, abnormal glycolipid metabolism, and neural network damage.

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