High-temperature skin thermal injury organ-like model and construction method thereof

Abstract

The invention provides a high-temperature skin thermal injury organ-like model and a construction method and application thereof. The high-temperature skin thermal injury organoid model is obtained by culturing a skin organoid obtained by differentiating mouse embryonic stem cells in a high-temperature environment. According to the invention, the skin organ injury model caused by high temperature is obtained by culturing the skin organ model in a high-temperature environment for the first time. Researches find that the model shows typical characteristics of activity reduction, heat stress, disorder of an ion transport system and the like. Therefore, the model can be used for research on a high-temperature skin heat injury mechanism and screening research on prevention and treatment targets.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue engineering, and specifically, to an organoid model of high-temperature skin thermal injury, a construction method thereof, and an application thereof. Background Art

[0002] The skin is an important physiological barrier for the body to resist external environmental damage. The impact of global warming and extreme high temperatures in special environments on skin health has become a global concern. High-temperature stress conditions can cause the reduction and denaturation of enzymes and functional proteins in the skin, accelerating the aging process of the skin. In addition, the increase in temperature exacerbates the skin inflammatory response, leading to skin oxidative damage. Therefore, establishing a pathological model of high-temperature-induced skin thermal injury is of great significance for clarifying the injury mechanism caused by environmental heat stress, developing targeted treatment means, better coping with the challenges of high-temperature climates, and ensuring long-term skin health.

[0003] Currently, the research models for high-temperature environment-induced skin thermal injury include in vitro models and in vivo models. The in vitro models mainly rely on two-dimensional culturing of one or more skin functional cells in a high-temperature environment. However, such models cannot fully simulate the real response of skin tissues due to the lack of a three-dimensional structure. The in vivo models mainly use animal models in a high-temperature breeding environment. However, animal models are often costly and cannot be carried out in large quantities, and have limitations in high-throughput mechanism research and drug screening.

[0004] With the continuous development of biotechnology, organoids, as an emerging biological model, have shown great potential in the fields of biomedical research, drug screening, etc. Different from the past skin three-dimensional models with only simple stratified structures or single appendages, the skin organoids constructed by Lee et al. through the differentiation of induced pluripotent stem cells can present an almost complete in vitro self-organizing stratified skin structure, and include appendages such as hair follicles and neural networks, which are ideal tools for in vitro modeling.

[0005] However, there is no report in the prior art on an organoid model with high biomimetic degree and applied to the research of high-temperature environment-induced skin damage, which will provide new ideas for the development of the organoid construction technology field. Summary of the Invention

[0006] To solve the above problems, the present invention provides an organoid model of high-temperature skin thermal injury, a construction method thereof, and an application thereof. The organoid model of high-temperature skin thermal injury is obtained by culturing skin organoids differentiated from mouse embryonic stem cells in a high-temperature environment, and has typical characteristics such as decreased activity, heat stress, and disorder of the ion transport system, with high biomimetic degree, aiming to solve the problems of lack of in vitro models, high cost of animal models, and inability to carry out high-throughput in the existing skin thermal injury research.

[0007] To achieve the above object, in a first aspect, the present invention provides a method for constructing a high-temperature skin thermal injury organoid model, comprising the following steps:

[0008] Embryonic stem cell maintenance culture step:

[0009] The mouse embryonic stem cells are passaged using LIF-2i medium, and the cell passage rate is maintained at 70-80%;

[0010] Skin organoid induction and differentiation step:

[0011] The mouse embryonic stem cells at passages 10-30 after passage are digested;

[0012] The digested mouse embryonic stem cells are resuspended in the first differentiation medium to form resuspended cells, and the resuspended cells are seeded on a 96-well low-cell adhesion U-bottom plate;

[0013] Cell aggregates are successively induced in each well using the second differentiation medium, the third differentiation medium, and the fourth differentiation medium;

[0014] Each formed cell aggregate is transferred to a well on a 24-well low-cell adhesion bottom plate in a maturation medium containing 1-5% (v / v) Matrigel. Half of the maturation medium is removed every other day, and the maturation medium without Matrigel is supplemented and cultured for 10-20 days to form skin organoids;

[0015] The components of the first differentiation medium include: GMEM medium, 1-2% (v / v) Knockout TM serum replacement, 1×MEM non-essential amino acid solution, 1-5 mM sodium pyruvate, 0.1-0.5 mM 2-mercaptoethanol, and 80-120 μg / ml Normocin;

[0016] The second differentiation medium is the first differentiation medium supplemented with 2-6% (v / v) Matrigel;

[0017] The third differentiation medium is the first differentiation medium supplemented with 30-70 ng / mL BMP-4 and 2-8 μM SB431542;

[0018] The fourth differentiation medium is the first differentiation medium supplemented with 3-10 μM LDN and 120-180 ng / mL FGF-2;

[0019] The components of the maturation medium include: Advanced DMEM / F12 medium, 1×N2 supplement, 1×GlutaMAX supplement, and 80-120 μg / ml Normocin;

[0020] Steps for high-temperature treatment of skin organoids:

[0021] Place mature skin organoids in a culture environment of 39 - 45°C and 5% CO2 for 42 - 50 h to obtain a high-temperature skin thermal injury organoid model. Preferably, the culture temperature is 40°C and the time is 46 - 48 h.

[0022] As a further improvement, during the high-temperature treatment of skin organoids, the cck-8 reagent is used to detect the activity of skin organoids during the high-temperature treatment process. When the culture time is 6 h, its activity increases.

[0023] As a further improvement, the LIF-2i medium is composed of DMEM / F12 and Neurobasal Medium mixed in a ratio of (1 - 5):(1 - 5), 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, 1 - 3 M PD0325901, 2 - 4 M CHIR99021, and 90 - 110 g / ml Normocin.

[0024] As a further improvement, the components of the first differentiation medium include: GMEM medium, 1.5 - 2% (v / v) Knockout TM serum replacement, 1× MEM non-essential amino acid solution, 1 - 3 mM sodium pyruvate, 0.1 - 0.3 mM 2-mercaptoethanol, and 90 - 110 g / ml Normocin;

[0025] The second differentiation medium is the first differentiation medium supplemented with 3 - 5% (v / v) Matrigel;

[0026] The third differentiation medium is the first differentiation medium supplemented with 40 - 60 ng / mL BMP-4 and 3 - 6 μM SB431542;

[0027] The fourth differentiation medium is the first differentiation medium supplemented with 4 - 7 μM LDN and 140 - 160 ng / mL FGF-2;

[0028] The components of the mature medium include: Advanced DMEM / F12 medium, 1× N2 supplement, 1× GlutaMAX supplement, and 90 - 110 g / ml Normocin.

[0029] As a further improvement, in the step of inducing and differentiating the skin organoids, mouse embryonic stem cells of the 18th to 20th passage are digested and resuspended. On the first day, 40 - 60 μL of the first differentiation medium is removed from each well and supplemented with 40 - 60 μL of the second differentiation medium, with a final concentration of 1 - 2%. On the third day, 20 - 30 μL of the third differentiation medium is added to each well to make the final volume 120 - 125 μL / well. On the fourth day, 20 - 30 μL of the fourth differentiation medium is added to each well to make the final volume 145 - 160 μL / well. On the eighth day, each cell aggregate is transferred into 450 - 550 μL of a maturation medium containing 1 - 2% (v / v) Matrigel, and from the tenth day, it is cultured in a maturation medium without Matrigel for 18 - 20 days.

[0030] As a further improvement, the final concentrations of BMP-4 and SB431542 in the third differentiation medium are 8 - 11 ng / mL and 1 - 3 μM respectively, and the final concentrations of LDN and FGF-2 in the fourth differentiation medium are 1 - 3 μM and 20 - 30 ng / mL respectively.

[0031] In a second aspect, there is provided a high-temperature skin thermal injury organoid model obtained from the first aspect above.

[0032] As a further improvement, the high-temperature skin thermal injury organoid model has differentially expressed genes, and the differentially expressed genes include heat stress-related genes.

[0033] As a further improvement, the heat stress-related genes of the high-temperature skin thermal injury organoid model include heat shock protein family A member 1A (Hspa1a) and heat shock protein family A member 1B (Hspa1b).

[0034] In a third aspect, there is provided the application of the high-temperature skin thermal injury organoid model of the first aspect or the second aspect in the research such as the mechanism of high-temperature skin thermal injury and the screening of prevention and treatment targets.

[0035] The above technical solutions of the present invention have the following beneficial effects:

[0036] (1) The high-temperature skin thermal injury organoid model of the present invention has typical characteristics such as decreased activity, heat stress, and disorder of the ion transport system, with high biomimetic degree.

[0037] (2) The method for constructing the high-temperature skin thermal injury organoid model of the present invention is simple, has good repeatability, and can meet the requirements of high-throughput and uniform model construction;

[0038] (3) The high-temperature skin thermal injury organoid model of the present invention can adjust the culture time according to the requirements of the lesion degree, and can meet the needs of personalized model customization.

[0039] (4) The high-temperature skin thermal injury organoid model of the present invention can be used for research such as the mechanism of skin thermal injury and the screening of prevention and treatment targets, and has broad application prospects. Description of the Drawings

[0040] Figure 1 : It is a flowchart for preparing an organoid model for studying high-temperature skin thermal injury of the present invention;

[0041] Figure 2 : It is a detection graph of the relative activity of the high-temperature skin thermal injury organoid model constructed by the present invention;

[0042] Figure 3 : It is an analysis of the differential gene expression before and after injury of the high-temperature skin thermal injury organoid model constructed by the present invention; where Organoid is a normal skin organoid, and Organoid+HT (High temperature) is a skin organoid model under high-temperature treatment; a. PCA graph, b. volcano graph;

[0043] Figure 4 : It is a heat map of the 10 genes with the most obvious differences among the up-regulated genes before and after injury of the high-temperature skin thermal injury organoid model constructed by the present invention;

[0044] Figure 5 : It is a GO analysis graph of biological processes in the enrichment analysis of differentially expressed genes before and after injury of the high-temperature skin thermal injury organoid model constructed by the present invention;

[0045] Figure 6 : It is a GO analysis graph of molecular functions in the enrichment analysis of differentially expressed genes before and after injury of the high-temperature skin thermal injury organoid model constructed by the present invention;

[0046] Figure 7 : It is to screen the key signal pathways and targets of injury using the differentially expressed genes before and after injury of the high-temperature skin thermal injury organoid model constructed by the present invention; a, screening key targets through a protein interaction network, b, expression levels of key target genes before and after high-temperature treatment. Detailed Embodiments

[0047] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention shall fall within the scope of the present invention.

[0048] Sources of reagents, culture media, kits, instruments or consumables, etc.:

[0049] R1 mouse embryonic stem cells (mESCs) (Cyagen Biosciences, MUAES-01001); TrypLE Express Enzyme (1X), no phenol red, Thermo Fisher; Matrigel (cultured at -20 degrees, a cell culture and differentiation reagent from Biofroxx, Germany); 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); TRIzol (Thermo Fisher, 15596018); NanoDrop ND-1000 Microvolume Spectrophotometer (NanoDrop, Wilmington, DE, USA); Agilent Bioanalyzer 2100 (Agilent, CA, USA); oligo(dT) magnetic beads (Dynabeads Oligo(dT), cat.25-61005, Thermo Fisher, USA); Magnesium RNA Fragmentation Module (NEBNext 1 MagnesiumRNA Fragmentation Module, cat.E6150S, USA); Reverse Transcriptase (InvitrogenTM 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 (Thermo Fisher, cat.R0133, CA, USA); UDG enzyme (Uracil-DNA Glycosylase) (NEB, cat.m0280, MA, US); illumina NovaseqTM 6000 gene sequencer (Hangzhou LC Sciences Co., Ltd.)

[0050] 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 Serum Replacement (Gibco); MEM Non-Essential Amino Acid Solution 11140050 (Gibco); Sodium Pyruvate (Gibco); 2-Mercaptoethanol (Gibco); Normocin Antimicrobial Reagent (Invivogen); Advanced DMEM / F12 Medium (Gibco); N2 Supplement (Gibco); GlutaMAX TM Supplement (Gibco);

[0051] 6-well clear 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).

[0052] Term Explanation:

[0053] 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 Technologies for evaluating the integrity of RNA, with a range of 1 - 10, where 1 represents the most severely degraded RNA and 10 represents the most intact RNA. The RIN value is calculated when detecting total eukaryotic RNA using an Agilent 2100 Bioanalyzer. "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 medium contains the supplement at a concentration of 1000 ug / ml. "Final concentration" refers to the final concentration of the solute in the medium.

[0054] The following will describe the embodiments of the present invention in detail in conjunction with the accompanying drawings and examples.

[0055] In some embodiments, the high-temperature skin thermal injury organoid model of the present invention is obtained by culturing skin organoids differentiated from mouse embryonic stem cells (mESCs) in a high-temperature environment. The "high-temperature environment" includes, but is not limited to, culturing the skin organoids in a high-temperature maturation medium for high-temperature culture.

[0056] In some embodiments, the high-temperature skin thermal injury organoid model of the present invention exhibits typical characteristics of skin thermal injury lesions such as decreased activity, heat stress, and disorder of the ion transport system. The "typical characteristics" may include abnormal expression levels of heat stress-related genes (heat shock protein family A member 1A (Hspa1a) and heat shock protein family A member 1B (Hspa1b)) different from normal levels.

[0057] In some embodiments, referring to Figure 1 , the method for constructing a high-temperature skin thermal injury organoid model includes the following steps:

[0058] It includes the following steps:

[0059] Embryonic stem cell maintenance culture step:

[0060] The mouse embryonic stem cells are passaged by culturing in LIF-2i medium, and the cell passage rate is maintained at 70-80%;

[0061] Skin organoid induction and differentiation step:

[0062] The mouse embryonic stem cells of the 10th, 15th, 20th, 25th, and 30th passages after passage are digested;

[0063] The digested mouse embryonic stem cells are resuspended in the first differentiation medium to form resuspended cells, and the resuspended cells are seeded on a 96-well low-cell adhesion U-bottom plate;

[0064] The cell aggregates are successively induced in each well using the second differentiation medium, the third differentiation medium, and the fourth differentiation medium;

[0065] Each formed cell aggregate is transferred to a well on a 24-well low-cell adhesion bottom plate in a maturation medium containing 1-5% (v / v) Matrigel. Half of the maturation medium is removed every other day, and the maturation medium without Matrigel is supplemented and cultured for 10-20 days to form skin organoids;

[0066] The components of the first differentiation medium include: GMEM medium, 1-2% (v / v) Knockout TMSerum substitute (wherein, "v / v" represents the volume ratio of Knockout Serum Substitute to GMEM medium, the same hereinafter), 1×MEM non-essential amino acid solution, 1-5 mM sodium pyruvate, 0.1-0.5 mM 2-mercaptoethanol, and 80-120 μg / ml Normocin;

[0067] The second differentiation medium is the first differentiation medium supplemented with 2-6% (v / v) Matrigel (wherein, "v / v" represents the volume ratio of Matrigel to "maturation medium", the same hereinafter). In a further technical solution, the "first differentiation medium containing Matrigel" contains 2, 3, 4, 5, or 6% (v / v) of Matrigel;

[0068] The third differentiation medium is the first differentiation medium supplemented with 30-70 ng / mL BMP-4 and 2-8 μM SB431542. In a further technical solution, the "first differentiation medium containing 5 μM BMP-4 and SB431542" contains 30, 35, 40, 45, 50, 55, 60, 65, or 70 ng / mL BMP-4 and 2, 3, 4, 5, 6, 7, or 8 μM of SB431542;

[0069] The fourth differentiation medium is the first differentiation medium supplemented with 3-10 μM LDN and 120-180 ng / mL FGF-2;

[0070] The components of the maturation medium include: Advanced DMEM / F12 medium, 1×N2 supplement, 1×GlutaMAX supplement, and 80-120 μg / ml Normocin; In these technical solutions, the "maturation medium" is not specifically limited, and known basal media are within the selection scope of the present invention. In a further technical solution, the "maturation medium containing Matrigel" contains 2-6% (v / v) of Matrigel (wherein, "v / v" represents the volume ratio of Matrigel to "maturation medium", the same hereinafter). In a further technical solution, the "maturation medium containing Matrigel" contains 2, 3, 4, 5, or 6% (v / v) of Matrigel.

[0071] Example 1 Construction of a high-temperature skin thermal injury organoid model

[0072] (1) Maintenance culture of embryonic stem cells (ESCs): Using a commercially purchased mouse R1 cell line, R1 mouse embryonic stem cells (mESCs) were cultured in LIF-2i medium without feeder cells. The cells were cultured on 6-well clear plates coated with 0.1% (w / v) gelatin, and the medium was changed every other day. The cell passage rate was maintained at 80%. mESCs at passage 30 were used for subsequent experiments.

[0073] (2) Inductive differentiation of skin organoids: On day 0, ESCs were dissociated with 1× TrypLE digestive enzyme, resuspended in the first 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).

[0074] On day 1, 50 μL of the medium was removed from each well and supplemented with 50 μL of the second differentiation medium containing 4% (v / v) Matrigel (final concentration 2%, Corning).

[0075] On day 3, 25 μL of the third differentiation medium, which is the first differentiation medium containing 50 ng / mL BMP-4 and 5 μM SB431542, was added to each well to make the final volume 125 μL / well. The final concentrations of BMP-4 and SB431542 were 10 ng / mL and 1 μM, respectively.

[0076] On day 4, 25 μL of the fourth differentiation medium, which is the first differentiation medium containing 6 μM LDN and 150 ng / mL FGF-2, was added to each well to make the final volume 150 μL / well. The final concentrations of LDN and FGF-2 were 1 μM and 25 ng / mL, respectively.

[0077] 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 the maturation medium containing 1% (v / v) Matrigel.

[0078] Starting from day 10, half of the medium (250 μL) was removed every other day and supplemented with 250 μL of the maturation medium without Matrigel until day 30.

[0079] (3) High-temperature treatment of skin organoids: On day 30, the mature skin organoids were cultured in an environment of 40 °C and 5% CO2 until day 32.

[0080] The LIF-2i medium is composed of a 1:1 mixture of DMEM / F12 and Neurobasal Medium, 0.5×N2 supplement, 0.5×B-27 supplement, 1×GlutaMAX supplement, 1000 U / ml leukemia inhibitory factor, 1 μM PD0325901, 3 μM CHIR99021, and 100 μg / ml Normocin.

[0081] The components of the first differentiation medium include: GMEM medium, 1.5% (v / v) Knockout TM serum replacement, 1×MEM non-essential amino acid solution, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, and 100 μg / ml Normocin;

[0082] The components of the maturation medium include: Advanced DMEM / F12 medium, 1×N2 supplement, 1×GlutaMAX supplement, and 100 μg / ml Normocin.

[0083] Control Example 1 constructs an organoid model without high-temperature skin thermal injury

[0084] Different from Example 1, the same skin organoids obtained in the example were cultured in the maturation medium.

[0085] Starting from the 30th day of Example 1, half of the maturation medium (250 μL) was removed every other day and supplemented with 250 μL of the maturation medium until the 32nd day of Example 1 to obtain an organoid model without high-temperature skin thermal injury.

[0086] Test Example 1 Detection of skin organoid activity

[0087] Test method: When starting the step of high-temperature treatment of skin organoids in Example 1, the activity of skin organoids during the high-temperature treatment was detected using the cck-8 reagent in accordance with the instructions at 37 / 40 / 42 / 45 °C at the 2 / 6 / 12 / 24 / 36 / 48 h, respectively, and comparisons were made, with 3 replicates for each time point. The detection results are as Figure 2 shown;

[0088] Analysis of test results: The control group could basically maintain stable activity in a 37°C culture environment; when treated at 40°C, the activity of skin organoids showed a stress-induced increase within 6 hours, and then gradually decreased. There was a significant difference from the control group at 24 hours. At the end of the 48-hour treatment, the activity could be maintained at about 60% of the pre-treatment level. This change conforms to the trend of human organs under stress conditions; when treated at 42°C or 45°C, the activity of skin organoids rapidly decreased within a short time and was basically completely inactivated at the end of the 48-hour treatment. Considering the degree of biomimetics and the stability of the model, a high-temperature skin thermal injury organoid model was constructed under the condition of 40°C.

[0089] Test Example 2: Test on changes in the expression levels of genes related to energy metabolism and thermal injury

[0090] Test group: Use the high-temperature skin thermal injury organoid model of Example 1, with 3 samples in each group;

[0091] Comparison group: The non-high-temperature skin thermal injury organoid model of Control Example 1, with 3 samples in each group;

[0092] Test method: Collect the test group and the comparison group, and use TRIzol (Thermo Fisher, 15596018) to isolate and purify the RNA of the total samples 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.

[0093] Use oligo(dT) magnetic beads (Dynabeads Oligo(dT), cat. 25-61005, Thermo Fisher, USA) to specifically capture the mRNA with PolyA (polyadenylic acid) through two rounds of purification. Fragment the captured mRNA under high-temperature conditions using a magnesium ion fragmentation kit (NEBNext® Magnesium RNA Fragmentation Module, cat. E6150S, USA) at 94°C for 5 - 7 minutes. Synthesize cDNA from the fragmented RNA through the action of reverse transcriptase (Invitrogen SuperScript™ II Reverse Transcriptase, cat. 1896649, CA, USA).

[0094] 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 these DNA-RNA hybrid double-strands into DNA double-strands. 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 end to enable ligation with adapters with T bases at the ends. Magnetic beads were used to screen and purify the fragment sizes.

[0095] 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 300 bp ± 50 bp.

[0096] Finally, Illumina NovaseqTM 6000 (LC Bio Technology CO., Ltd., Hangzhou, China) was used to perform paired-end sequencing on it according to the standard operation, and the sequencing mode was PE150. After using Cutadapt to filter out unqualified sequences (sequencing adapters, low-quality sequences, etc.) in the raw data to obtain valid data (Clean Data), Hisat2 was used for reference genome alignment. According to the alignment results of Hisat2, StringTie was used to reconstruct transcripts and calculate the expression levels of all genes in each sample.

[0097] The gene expression level analysis mainly targeted 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.

[0098] When measuring the gene expression level, the FPKM value (Fragments Per Kilobase Million, standardized from 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. The threshold criteria for screening differentially expressed genes were 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) (|log2FC| >= 1 & q < 0.05). In the set comparison groups, differentially expressed genes were obtained and enrichment analysis was performed, focusing on the changes in the expression levels of genes related to energy metabolism and heat damage.

[0099] See Figure 3 The results of principal component analysis Figure 3 showed good intra-group repeatability in the experimental group and the control group, indicating the stability of the model. The significant inter-group differences indicated that the model successfully induced changes in the gene expression pattern of skin organoids (a); compared with the control group, there were 924 up-regulated genes and 2,109 down-regulated genes in the experimental group (b). (HT2 represents the experimental group, i.e., high temperature treatment for 2 days, and NC2 represents the control group, i.e., negative control treatment for 2 days);

[0100] See Figure 4 Figure 4 , further research on the genes with the most significant differential expression in the differential expression analysis found that heat stress-related genes such as heat shock protein family A member 1A (Hspa1a) and heat shock protein family A member 1B (Hspa1b) appeared within the top 10 genes with the most significant differences, indicating that the model exhibited typical characteristics of heat stress during heat injury and the model construction was successful;

[0101] See Figure 5 and Figure 6 Figure 5 and Figure 6 , the differential genes between the experimental group and the control group were subjected to GO analysis, focusing on two aspects: biological process and molecular function; in terms of biological process, multiple entries related to the transport of single ions such as potassium ions and transmembrane transport were enriched ( Figure 5 ); in terms of molecular function, multiple entries related to the transport of single ions such as potassium and calcium and ion binding were enriched ( Figure 6 ); these changes were consistent with the dysfunction of cell membrane ion channels and pumps and the imbalance of intracellular ion homeostasis during high-temperature skin injury, which in turn caused barrier function damage and tissue repair disorders, proving that the model was consistent with the pathophysiological changes during skin heat injury.

[0102] Based on the test results of Test Examples 1 and 2 above, the high-temperature skin heat injury organoid model of the present invention has typical characteristics of skin heat injury lesions such as decreased activity, heat stress, and disorder of the ion transport system, and has a high degree of biomimesis.

[0103] Test Example 3: Screening of prevention and treatment targets

[0104] To explore the targets for improving skin high-temperature tolerance, the top 20 ion transport system-related entries with the highest enrichment significance in the GO analysis were screened, and protein network interaction analysis was performed on the genes with an enrichment frequency of 4 or more times among the genes included in the entries. The results showed ( Figure 7) The α1 subunit of the sodium channel (Scn1a) gene has a strong association with other genes, and the degree of association is higher, indicating its important role in maintaining the homeostasis of the ion transport system at high temperatures (a); at the same time, gene expression analysis found that the expression of this gene decreased to about 20% of the normal level at high temperatures (b). Therefore, increasing the expression level of this gene and enhancing the stability of the protein it encodes are potential targets for preventing and treating high-temperature thermal injury.

[0105] The embodiments of the present invention also have the following beneficial effects:

[0106] (1) The method for constructing the high-temperature skin thermal injury organoid model of the present invention is simple, has good repeatability, and can meet the requirements for constructing high-throughput and homogeneous models;

[0107] (2) Referring to Figure 2 , when selecting different temperatures such as 42 / 45 degrees, 24 hours or other time points when the organoids are still active can be selected as the end time to construct the model. The damage degrees under different conditions are different, indicating that the high-temperature skin thermal injury organoid model of the present invention can adjust the culture time according to the requirements of the lesion degree and can meet the needs of personalized model customization;

[0108] (3) The high-temperature skin thermal injury organoid model of the present invention can be used for research such as the mechanism of high-temperature skin thermal injury and the screening of prevention and treatment targets, and has broad application prospects;

[0109] (4) The high-temperature skin thermal injury organoid model of the present invention fills the gap in the in vitro three-dimensional model of skin thermal injury, reduces the cost of in vivo animal experiments, and avoids potential ethical issues.

[0110] The above-described embodiments are only described as the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, 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 method for constructing a high-temperature skin thermal injury organoid model, characterized in that: It includes the following steps: Embryonic stem cell maintenance culture step: The mouse embryonic stem cells are passaged by culturing them in LIF-2i medium, and the cell passage rate is maintained at 70-80%; Skin organoid induction and differentiation step: The mouse embryonic stem cells at passages 10-30 after passage are digested; The digested mouse embryonic stem cells are resuspended in the first differentiation medium to form resuspended cells, and the resuspended cells are seeded on a 96-well low-cell adhesion U-bottom plate; Cell aggregates are successively induced in each well using the second differentiation medium, the third differentiation medium, and the fourth differentiation medium; Each formed cell aggregate is transferred to a well on a 24-well low-cell adhesion bottom plate in a maturation medium containing 1-5% (v / v) Matrigel. Half of the maturation medium is removed every other day, and the maturation medium without Matrigel is supplemented and cultured for 10-20 days to form skin organoids; The components of the first differentiation medium include: GMEM medium, 1-2% (v / v) Knockout TM serum substitute, 1×MEM non-essential amino acid solution, 1-5 mM sodium pyruvate, 0.1-0.5 mM 2-mercaptoethanol, and 80-120 μg / ml Normocin; The second differentiation medium is the first differentiation medium supplemented with 2-6% (v / v) Matrigel; The third differentiation medium is the first differentiation medium supplemented with 30-70 ng / mL BMP-4 and 2-8 μM SB431542; The fourth differentiation medium is the first differentiation medium supplemented with 3-10 μM LDN and 120-180 ng / mL FGF-2; The components of the maturation medium include: Advanced DMEM / F12 medium, 1×N2 supplement, 1×GlutaMAX supplement, and 80-120 μg / ml Normocin; High-temperature treatment of skin organoids step: The mature skin organoids are cultured in a 39-45°C, 5% CO2 culture environment for 42h-50h to obtain a high-temperature skin thermal injury organoid model. Preferably, the culture temperature is 40°C and the time is 46-48h.

2. The method for constructing a high-temperature skin thermal injury organoid model according to claim 1, wherein In the high-temperature treatment of skin organoids step, the skin organoids during the high-temperature treatment are detected for activity using cck-8 reagent, and when the culture time is 6h, its activity increases.

3. The method for constructing a high-temperature skin thermal injury organoid model according to claim 1, wherein: The LIF-2i medium is composed of DMEM / F12 and Neurobasal Medium mixed in a ratio of (1-5):(1-5), 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, 1-3 μM PD0325901, 2-4 μM CHIR99021, and 90-110 μg / ml Normocin.

4. The method for constructing a high-temperature skin thermal injury organoid model according to claim 1, characterized in that: The components of the first differentiation medium include: GMEM medium, 1.5-2% (v / v) Knockout TM serum substitute, 1×MEM non-essential amino acid solution, 1-3 mM sodium pyruvate, 0.1-0.3 mM 2-mercaptoethanol, and 90-110 μg / ml Normocin; The second differentiation medium is the first differentiation medium supplemented with 3-5% (v / v) Matrigel; The third differentiation medium is the first differentiation medium supplemented with 40-60 ng / mL BMP-4 and 3-6 μM SB431542; The fourth differentiation medium is the first differentiation medium supplemented with 4-7 μM LDN and 140-160 ng / mL FGF-2; The components of the maturation medium include: Advanced DMEM / F12 medium, 1×N2 supplement, 1×GlutaMAX supplement, and 90 - 110 μg / ml Normocin.

5. The method for constructing a high-temperature skin thermal injury organoid model according to claim 1, wherein: In the step of inducing and differentiating skin organoids, mouse embryonic stem cells of passage 18 - 20 are digested and resuspended. On the first day, 40 - 60 μL of the first differentiation medium is removed from each well and supplemented with 40 - 60 μL of the second differentiation medium, with a final concentration of 1 - 2%. On the third day, 20 - 30 μL of the third differentiation medium is added to each well to make the final volume 120 - 125 μL / well. On the fourth day, 20 - 30 μL of the fourth differentiation medium is added to each well to make the final volume 145 - 160 μL / well. On the eighth day, each cell aggregate is transferred into 450 - 550 μL of the maturation medium containing 1 - 2% (v / v) Matrigel, and starting from the tenth day, it is cultured in the maturation medium without Matrigel for 18 - 20 days.

6. The method for constructing a high-temperature skin thermal injury organoid model according to claim 1, characterized in that: The final concentrations of BMP-4 and SB431542 in the third differentiation medium are 8 - 11 ng / mL and 1 - 3 μM respectively, and the final concentrations of LDN and FGF-2 in the fourth differentiation medium are 1 - 3 μM and 20 - 30 ng / mL respectively.

7. An organoid model of high-temperature skin thermal injury is obtained by using the construction method according to any one of claims 1 - 6.

8. The high-temperature skin thermal injury organoid model according to claim 7, characterized in that, The organoid model of high-temperature skin thermal injury has differentially expressed genes, and the differentially expressed genes include heat stress-related genes.

9. The high-temperature skin thermal injury organoid model according to claim 8, wherein The heat stress-related genes of the organoid model of high-temperature skin thermal injury include heat shock protein family A member 1A (Hspa1a) and heat shock protein family A member 1B (Hspa1b).

10. An application of the organoid model of high-temperature skin thermal injury according to any one of claims 1 - 9 in the research such as the mechanism of high-temperature skin thermal injury and the screening of prevention and treatment targets.

Images