Method for inducing an animal model of type 1 diabetes with antigen-specific t cells

Abstract

The present application provides a preparation method of a non-human mammal model of type 1 diabetes. Specifically, the present application induces islet beta cell destruction in a non-immunodeficient mouse by using T cells specifically targeting GAD, constructs T cell autoimmune type 1 diabetes in line with the actual pathogenesis, and has application prospects in the field of diabetes research.

Description

Technical Field

[0001] This invention relates to the field of biological models. Specifically, this invention relates to a method for establishing an antigen-specific T cell-induced type 1 diabetes animal model. Background Technology

[0002] Type 1 diabetes mellitus (T1DM) is an organ-specific autoimmune disease characterized by inflammatory infiltration of the pancreatic islets and the continuous destruction of pancreatic β cells by autoreactive immune cells, especially T cells. This results in insufficient insulin production to maintain glucose homeostasis, leading to symptoms such as hyperglycemia. Animal models of T1DM have significantly advanced research into its pathogenesis and drug development. Commonly used T1DM research models include the NOD mouse model and chemical injections such as streptozotocin (STZ) or alloxan to induce T1DM. The T1DM phenotype in NOD mice is caused by a genetic defect, exhibiting multiple immunophenotypic abnormalities, including deficiencies in antigen-presenting cell immune regulation, T lymphocyte regulation, and NK cell function. Therefore, it cannot accurately simulate the pathology of T1DM individuals. In contrast, STZ and other drug-induced T1DM models primarily result from the direct destruction of β cells by the chemical substances, rather than autoimmunity causing T1DM.

[0003] Therefore, in order to better promote related research and drug development, the field still needs to develop methods for constructing animal models of T1DM that can simulate the pathological process of T1DM in non-immune-deficient wild-type animals. Summary of the Invention

[0004] The purpose of this invention is to provide a method for constructing a wild-type T1DM animal model.

[0005] In a first aspect of the invention, a method for preparing a non-human mammalian model of type 1 diabetes is provided, wherein the animal is a non-immunodeficient animal, and the method comprises administering T cells specifically targeting GAD to the animal.

[0006] In another preferred embodiment, the animal is a rodent or primate, preferably including a mouse, rat, rabbit, or monkey.

[0007] In another preferred embodiment, the animal is a C57BL / 6 mouse.

[0008] In another preferred embodiment, the T cells are prepared by the following method:

[0009] A population of cells is provided that has been immune-stimulated with GAD antigen peptides, from which T cells are isolated.

[0010] In another preferred embodiment, the immune stimulation includes in vivo immune stimulation or in vitro immune stimulation.

[0011] In another preferred embodiment, the immune stimulation comprises stimulation with a GAD antigen peptide having an amino acid sequence such as AFLHATDLL (SEQ ID NO. 1) or YAFLHATDL (SEQ ID NO. 2).

[0012] In another preferred embodiment, the T cells that specifically target GAD specifically bind to an antigenic epitope peptide such as AFLHATDLL (SEQ ID NO. 1).

[0013] In another preferred embodiment, the T cells are CD4 cells. + T cells, CD8 + T cells, or combinations thereof.

[0014] In another preferred embodiment, the T cells are CD8. + T cells.

[0015] In another preferred embodiment, the amount of T cells used does not exceed 5 × 10^6 cells / animal, preferably not more than 3 × 10^6 cells / animal, and more preferably not more than 1 × 10^6 cells / animal.

[0016] In another preferred embodiment, the method further includes administering a compound selected from the group consisting of:

[0017] STZ, alloxan, or combinations thereof.

[0018] In another preferred embodiment, the total amount of STZ administered does not exceed 300 mg / kg body weight, preferably not more than 200 mg / kg body weight, more preferably not more than 150 mg / kg body weight, and even more preferably not more than 80 mg / kg body weight.

[0019] In another preferred embodiment, the single dose of the STZ does not exceed 80 mg / kg body weight, preferably not more than 60 mg / kg body weight, and more preferably not more than 40 mg / kg body weight.

[0020] In another preferred embodiment, the number of pancreatic β cells in the animal decreases, preferably by more than 50%, more preferably by more than 80%.

[0021] In another preferred embodiment, the decrease in the number of pancreatic β cells is caused by T cell-specific killing.

[0022] In a second aspect of the invention, the use of an animal model prepared by the method described in the first aspect of the invention is provided for studying the pathogenesis of T1DM.

[0023] In a third aspect of the invention, the use of an animal model prepared by the method described in the first aspect of the invention is provided for screening or identifying substances that can prevent or treat T1DM.

[0024] In a fourth aspect of the invention, a method for preparing GAD-specific T cells is provided, the method comprising the steps of:

[0025] 1) Provide a population of cells stimulated by GAD, and isolate a population of T cells from the population of cells;

[0026] 2) Isolate T cells from the T cell population that specifically bind to the antigenic peptide shown in SEQ ID NO.1.

[0027] In another preferred embodiment, the separation includes isolating GAD-specific T cells from the T cell population using an MHC-antigen peptide complex, wherein the antigen peptide sequence contains the sequence shown in SEQ ID NO.1.

[0028] In another preferred embodiment, the cell population can be obtained through in vivo or in vitro immune stimulation.

[0029] In another preferred embodiment, the immune stimulation comprises stimulation with a GAD antigen peptide having an amino acid sequence such as AFLHATDLL (SEQ ID NO. 1) or YAFLHATDL (SEQ ID NO. 2).

[0030] In another preferred embodiment, the T cells are CD8. + T cells.

[0031] In another preferred embodiment, the cell population is derived from animals or is cultured artificially.

[0032] In another preferred embodiment, the cell population is derived from animals selected from the group consisting of mice, rats, and humans.

[0033] In another preferred embodiment, the cell population is selected from the group consisting of peripheral blood mononuclear cells (PBMCs), splenic lymphocytes or single cells, pancreatic draining lymph cells, pancreatic immune infiltrating cells, or combinations thereof.

[0034] In a fifth aspect of the invention, a GAD-specific T cell is provided, said T cell being prepared by the method described in the fourth aspect of the invention.

[0035] In a sixth aspect of the invention, a method is provided for screening or identifying potential therapeutic agents that can prevent or treat T1DM, comprising the steps of:

[0036] (a) In the test group, in the presence of the test compound, the test compound is applied to an animal model prepared by the method described in the first aspect of the invention, and the blood glucose level G1 of the animal model in the test group is monitored; in the control group, in the absence of the test compound, a control compound (e.g., a solvent) is applied to an animal model prepared by the method described in the first aspect of the invention, and the blood glucose level G2 of the animal model in the test group is monitored.

[0037] (b) Compare the blood glucose values ​​G1 and G2 detected in the previous step to determine whether the test compound is a potential therapeutic agent for the prevention and / or treatment of T1DM;

[0038] If the blood glucose value G1 is significantly lower than the blood glucose value G2, it indicates that the test compound is a potential therapeutic agent for the prevention and / or treatment of T1DM.

[0039] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0040] In a seventh aspect of the invention, a T1DM model of a non-human mammal is provided, the model being prepared using the method described in the first aspect of the invention.

[0041] In an eighth aspect of the invention, a composition for preparing a T1DM nonhuman mammalian model is provided, the composition comprising:

[0042] M1) T cells that specifically target GAD; and

[0043] M2) Other substances that induce T1DM.

[0044] In another preferred embodiment, the M2 includes STZ, alloxan, or a combination thereof.

[0045] In another preferred embodiment, the T cells that specifically target GAD specifically bind to an antigenic epitope peptide such as AFLHATDLL (SEQ ID NO. 1).

[0046] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0047] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0048] Figure 1The blood glucose concentration curves of mice are shown. *p<0.05, ***p<0.001, ****p<0.0001; n=5.

[0049] Figure 2 Immunofluorescence staining reveals the β-cell content in the pancreas: (A) representative islets from the adoptive transfer (AT) immune cell group, and (B) representative islets from the PBS group. MAFA (green), DAPI (blue). Scale bar: 50 μm.

[0050] Figure 3 Immunofluorescence of the pancreas is shown. AT, representative islets from the adoptive-transfer immune cell group; PBS, representative islets from the injected PBS group. CD3 (green), glucagon (red), DAPI (blue). Scale bar: 50 μm.

[0051] Figure 4 The adoptive immunization flowchart of the present invention is shown. Detailed Implementation

[0052] Through extensive and in-depth research, the inventors have developed, for the first time, a method for establishing an antigen-specific T cell-induced type 1 diabetes animal model. Wild-type mice (e.g., C57BL / 6) are immunized with islet autoantigens to produce abundant islet autoantigen-specific T cells. After isolation and purification, these islet autoantigen-specific T cells are obtained and adoptively transferred to healthy mice of the same strain, inducing the mice to progress to type 1 diabetes (T1DM), thus constructing an antigen-specific T cell-induced autoimmune T1DM model. Based on this, the present invention was completed.

[0053] GAD

[0054] Glutamic acid decarboxylase (GAD) is the rate-limiting enzyme that catalyzes the conversion of glutamate to GABA, a major inhibitory neurotransmitter in the central nervous system. In addition to being found in all GABAergic neurons, GAD has also been detected in pancreatic islet cells.

[0055] In mammals, there are two isoforms of GAD: GAD65 and GAD67. While their structures are basically similar, their stereoconformities and antigenic epitopes differ. GAD65 is predominantly found in the pancreas, while GAD67 is predominantly found in brain tissue.

[0056] GAD65 full-length sequence (Mus musculus, mouse):

[0057] (SEQ ID NO.3)

[0058] This invention uses T cells that specifically target GAD to construct a T1DM model. The T cells of this invention can be any T cell that specifically binds to the GAD protein, such as T cells that specifically recognize the epitope sequence AFLHATDLL (SEQ ID NO.1, positions 90-98 of GAD65 protein).

[0059] The GAD-specific T cells used in this invention can be prepared using the following steps:

[0060] 1) Provide a population of cells stimulated by GAD, and isolate a population of T cells from the population of cells;

[0061] 2) GAD-specific T cells were isolated from the T cell population using an MHC-antigen peptide complex, wherein the antigen peptide sequence is shown in SEQ ID NO.1.

[0062] The T cells of the present invention can be derived from cell populations from the following sources: peripheral blood mononuclear cells (PBMCs), splenic lymphocytes or single cells, pancreatic draining lymphocytes, pancreatic immune infiltrating cells, or combinations thereof. The cell populations can be derived from animals or cultured artificially. The cell populations are derived from animals selected from the group consisting of mice, rats, and humans. The cell populations can be obtained through in vivo or in vitro immune stimulation, including stimulation with GAD antigen peptides whose amino acid sequences are represented as AFLHATDLL (SEQ ID NO. 1) or YAFLHATDL (SEQ ID NO. 2).

[0063] animal models

[0064] This invention provides a non-human mammalian model of type 1 diabetes mellitus (T1DM). The T1DM animal model of this invention is an autoimmune T1DM model induced by antigen-specific T cells.

[0065] In this invention, examples of non-human mammals include (but are not limited to): mice, rats, rabbits, monkeys, etc., more preferably rats and mice.

[0066] The method of this invention can establish T1DM models in non-immunodeficient non-human mammals, such as wild-type animals, preferably C57BL / 6 mice, but not limited thereto. It should be understood that the compositions and methods of this invention can also be used to establish T1DM models in immunodeficient animals; therefore, this invention can also be applied to the establishment of models in Kunming mice, BALB / C mice, NOD mice, and NOD / SCID mice, but is not limited thereto.

[0067] In one embodiment of the invention, the animal model of the invention is constructed by combined administration of M1) T cells specifically targeting GAD; and M2) other chemicals (such as STZ).

[0068] Through research, the inventors discovered that streptozotocin does not induce T1DM in non-immunodeficient mice when administered at low doses and low frequencies (e.g., total dose not exceeding 100 mg / kg), and the percentage of pancreatic β cells in mice remains normal after administration. However, when used in combination with GAD-specific T cells, streptozotocin can effectively induce pancreatic β cell destruction in non-immunodeficient mice, thus constructing a T1DM model that conforms to the actual pathogenesis.

[0069] The main advantages of this invention include:

[0070] 1) Compared with the NOD mouse model, the specific T cells of the present invention can induce the formation of an autoreactive T1DM model in mice with normal immune systems (such as C57BL / 6 mice), which makes up for the shortcomings of NOD mice in that they have immune deficiencies and cannot reflect the real pathology.

[0071] 2) This invention found that low-dose, low-frequency STZ administration alone does not cause disease, but can promote specific T cell-induced autoimmune responses, thereby destroying pancreatic β cells; this method is more consistent with the pathogenesis of T1DM compared with multiple low-dose chemical-induced β cell death by STZ alone.

[0072] 3) The modeling method of the present invention is conducive to better elucidating the pathogenesis of T1DM, promoting the search for targets and methods of T1DM immunotherapy, and screening drugs for the treatment of T1DM.

[0073] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0074] Experimental materials:

[0075] T-cell sorting kit (STEMCELL)

[0076] CD8 T Cell Sorting Kit (STEMCELL)

[0077] Isolation of MHC tetramers from antigen-specific T cells (Haozhi Biotechnology)

[0078] C57BL / 6 mice (Jicui Yaokang)

[0079] Immunorelubricant CFA (Chondrex)

[0080] Antigenic peptide (Sangon Biotech)

[0081] Blood glucose monitor (Roche)

[0082] OCT (Sakura)

[0083] 30% sucrose solution: Weigh 30g of sucrose, dissolve it in 1×PBS solution, and bring the volume to 100ml.

[0084] The anti-glucagon antibody (Abcam) was diluted at a ratio of 1:500.

[0085] anti-CD3 antibody (CST Corporation) dilution ratio 1:400.

[0086] MAFA antibody (CST) dilution ratio 1:400.

[0087] 0.5M citrate buffer, pH 4.5 (Beijing Tianenze)

[0088] Example 1: Immunization of animals and T-cell screening

[0089] 1.1 Preparation of Antigenic Peptide Emulsion

[0090] (1) The lyophilized antigen peptide is diluted with PBS to 2 mg / ml. Usually, each mouse needs 200 μg of GAD65 antigen peptide (take 0.1 ml of the diluted solution). The antigen peptide solution is stored at -20℃.

[0091] (2) The antigen peptide and CFA were injected into each mouse at a 1:1 ratio (100 μl each). Considering the loss of viscous emulsion during preparation and injection, prepare 1.5-2 times the required amount (e.g., 1 ml of antigen peptide and CFA for 10 mice; prepare 2 ml of each to prevent loss).

[0092] (3) Transfer the solvent from one syringe to another and mix thoroughly for at least 10 minutes. Good emulsification is a crucial step. Once emulsification is complete, close the three-way valve (the emulsion should be white, firm, and viscous, with no phase separation. When dropped into clean water, it should float on the surface without dispersing). After preparing the emulsion, wait at least 30 minutes to observe its stability.

[0093] 1.2 Animal Immunization

[0094] (1) Anesthetize the mice (assess the level of anesthesia by pinching the toes).

[0095] (2) 100 μl of antigen peptide emulsion was injected subcutaneously into two different sites on the posterior flank of the mouse (the needle remained in the body for 15 seconds after injection, and a spherical mass was observed to form under the skin of the mouse, which persisted throughout the experiment).

[0096] (3) Throughout the experiment, each mouse was marked by its tail and its blood glucose levels were recorded.

[0097] 1.3 Isolation and Extraction of T Cells

[0098] STEMCELL's EasySep TM The Mouse T Cell Isolation Kit was used to isolate T cells according to the instructions. The experimental steps are as follows:

[0099] (1) Adjust the concentration of the lymphocyte suspension to 1×10^8 cells / mL and take 1-1.5ml.

[0100] (2) Add rat serum at a ratio of 50 μl / ml and mix with a pipette.

[0101] (3) Add T Cell Isolation Cocktail at a ratio of 50 μl / ml, mix with a pipette, and incubate at room temperature for 10 min.

[0102] (4) Add 75 μl / ml of Streptavidin RapidSpheres TM Mix the beads well. Make up the volume to 2.5 ml with buffer (1×PBS+2% FBS), place on a magnetic rack, and incubate at room temperature for 2.5 min.

[0103] (5) Transfer the supernatant to a new flow cytometry tube (5 ml), place the flow cytometry tube containing the cell supernatant on a magnetic rack, and incubate at room temperature for 2.5 min.

[0104] (6) Collect the supernatant into a new centrifuge tube and centrifuge at 4°C and 1500 rpm for 10 min. Discard the supernatant.

[0105] (7) Add buffer (1×PBS+2% FBS) to resuspend the cells and collect the T cells, which are the T cell population obtained by GAD immunization.

[0106] 1.4 Antigen-specific T cell isolation

[0107] 1. Resuspend the collected T cells in flow cytometry buffer (FACS Buffer) at a density of 2-5 x 10^7 / ml to prepare a single-cell suspension. The FACS Buffer consists of 1×PBS + 2% fetal bovine serum + 0.1% sodium azide and should be prepared fresh before use. Add 25 μl of the cell suspension to a 5 ml FACS tube to obtain a T cell subset containing CD4 / CD8 T cells.

[0108] 2. Preparation of Staining Cocktail: Take 10 μl of MHC-antigen peptide tetramer, dilute it in FACS Buffer and dilute it 50 times. The sequence of the antigen peptide is shown in SEQ ID NO.1.

[0109] 3. Staining incubation: Take 25 μl of staining cocktail and add it to an FACS tube containing cell suspension. Gently pipette to mix, avoiding the formation of air bubbles as much as possible.

[0110] 4. Incubate on ice or in a 4°C refrigerator for 60 minutes in the dark.

[0111] 5. Washing: Add 150 μL of FACS Buffer to the FACS tube from step 4, mix gently to avoid forming air bubbles, centrifuge at 1200 rpm for 5 min, carefully remove the supernatant, and avoid touching the cell pellet to reduce cell loss.

[0112] 6. Repeat step 5, washing twice. Use magnetic beads containing PE-specific antibodies to sort PE-labeled tetramer-positive cells. The resulting positive cells are CD8 T cells.

[0113] Example 2: Adoptive transfer combined with STZ-induced T1DM

[0114] The inventors’ previous research confirmed that cell populations induced by GAD antigen peptide immunization alone could not induce T1DM in C57BL / 6 mice. Therefore, in this embodiment, STZ combined with T cell adoptive transfer therapy was used to induce T1DM.

[0115] 2.1 Mouse grouping and treatment

[0116] Ten approximately 8-week-old mice were randomly divided into an AT (Adoptive Transfer) group and a PBS group. Blood glucose levels were recorded as day 0. The two groups of mice were treated as follows:

[0117] AT group: 200 μl containing 5 × 10^6 T cells was transferred to C57BL / 6 mice via tail vein injection, and 40 mg / kg STZ solution was injected intraperitoneally on days 2 and 3, respectively.

[0118] PBS group: 200 μl of PBS solution was injected into C57BL / 6 mice via the tail vein, and 40 mg / kg of STZ solution was injected intraperitoneally on days 2 and 3.

[0119] 2.2 Blood glucose testing

[0120] The mouse was removed from its cage and gently placed on a wire mesh. Approximately 1-2 mm of the mouse's tail was trimmed with surgical scissors, and the tail was gently squeezed to concentrate the blood into a single drop. A Roche blood glucose monitor was then used to measure the mouse's blood glucose level.

[0121] Result: As Figure 1 As shown, after 17 days, mice in the AT group began to show symptoms of hyperglycemia, while the blood glucose level in the PBS group remained normal. Mice were considered to have progressed to type 1 diabetes mellitus (T1DM) when their blood glucose concentration was greater than 13.8 mmol / L for two consecutive days.

[0122] 2.3 Immunofluorescence staining

[0123] To further examine the presence of pancreatic β cells in mice with hyperglycemia and the pathological characteristics of type 1 diabetes mellitus (T1DM), this example uses pancreatic tissue immunofluorescence staining to detect pancreatic T cell infiltration and β cell survival. The method is as follows:

[0124] (1) After euthanizing the mice, they were soaked in 75% ethanol and the pancreatic tissue of the mice was taken from the ventral side.

[0125] (2) Fix 4% PFA overnight in a 4°C refrigerator.

[0126] (3) Subsequently, the pancreatic tissue was dehydrated with a 30% sucrose solution until it was completely settled in the solution.

[0127] (4) Remove the completely settled pancreatic tissue from the 30% sucrose solution and carefully blot dry the surface liquid on filter paper. Rinse once with 1×PBS solution and blot dry. Embed the pancreatic tissue in an OCT chamber. Place the embedded tissue in a 4℃ freezer for 15-30 minutes to remove air bubbles. Quick-freeze the embedded tissue in a -80℃ freezer for 30 minutes. Store the frozen tissue at -20℃.

[0128] (5) When slicing, take it out and fix it on the stage of the cryostat. Select the tissue to be sliced ​​and the slice thickness is 7μm.

[0129] (6) After the cut sections are air-dried at room temperature, rinse the tissue sections with 1×PBS solution for 5-10 min.

[0130] (7) Place the slide in 0.3% PBST solution for 30 min to break the membrane (1*PBS contains 0.3% Tritonx100. Tritonx100 is viscous, so pay attention to the pipette tip and usage when preparing it to prevent clogging).

[0131] (8) Shake off the residual liquid, wipe off the liquid around the tissue with a tissue, circle the slide with the tissue with a histochemistry pen, place it in a dark box and seal it at room temperature for 2 hours with 10% FBS.

[0132] (9) Discard the blocking solution from the sealed slide, wipe away excess liquid with a paper towel, and maintain the integrity of the circles on the histochemistry pen. Add anti-glucagon (Abcam), anti-CD3 (CST), and MAFA (CST) antibodies as primary antibodies, and incubate overnight at 4°C.

[0133] (10) Rinse tissue sections three times with 1×PBS for 10 min each time.

[0134] (11) Add a fluorescent secondary antibody of the same species as the primary antibody and incubate at 37°C in the dark for two hours.

[0135] (12) After incubation with the secondary antibody, rinse the tissue sections 5 times with 1×PBS in the dark for 7 minutes each time, and then air dry.

[0136] (13) Apply DAPI-containing sealing tablets to the tablet and seal it. Let it air dry in the dark.

[0137] (14) Fluorescence observation and analysis were performed on a laser confocal microscope. The corresponding laser channels (405, 488, and 633 nm, respectively) were selected according to the fluorescence secondary antibody for fluorescence observation and analysis.

[0138] Results: MAFA is a transcription factor specific to mouse pancreatic β-cells and is expressed in β-cells, serving as a β-cell marker. The presence of β-cells in mice was assessed by labeling β-cells with anti-MAFA. Normally, pancreatic β-cells comprise approximately 80% of all pancreatic islet cells in mice. The AT group showed significant loss of pancreatic β-cells, while the PBS group exhibited a normal percentage of β-cells. Figure 2 ).

[0139] The results indicate that the STZ dose used in this invention does not cause significant damage to pancreatic β cells in mice. Therefore, the constructed model is basically caused by T cell autoimmunity alone, which more realistically simulates the pathogenesis of T1DM.

[0140] 2.4 Pancreatic T-cell infiltration

[0141] T cells were labeled with anti-CD3 antibody to observe T cell infiltration in pancreatic islet tissue. It was found that no immune cell infiltration was observed in the pancreatic tissue of mice in the PBS group, while immune cell infiltration into the islet tissue was observed in the AT group, leading to pancreatitis. Figure 3 ).

[0142] It was observed that immune cell infiltration and β-cell loss were present in the tissues of T1DM mice. This indicates that the model meets the physiological (hyperglycemia) and pathological (insulitis) conditions for a T1DM model.

[0143] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for preparing a non-human mammalian model of type 1 diabetes, characterized in that, The animals are non-immunodeficient animals, and the method includes administering CD8, which specifically targets GAD, to the animals. + T cells and streptozotocin; The T cells that specifically target GAD specifically bind to the antigenic epitope peptide represented by AFLHATDLL (SEQ ID NO.1); And the animal in question is a mouse; The adoptive transfer of 200 μl containing 5 × 10^6 T cells was injected into mice via the tail vein, and on days 2 and 3, mice were intraperitoneally injected with 40 mg / kg streptozotocin solution.

2. The preparation method according to claim 1, characterized in that, The animal in question is a C57BL / 6 mouse.

3. The preparation method according to claim 1, characterized in that, The T cells were prepared by the following method: Provide a cell population that has been immune-stimulated with GAD antigen peptides, and isolate T cells from the cell population; The immune stimulation includes stimulation with a GAD antigen peptide with an amino acid sequence such as AFLHATDLL (SEQ ID NO.1).

4. The use of the animal model prepared by the method described in claim 1, characterized in that, Used to study the pathogenesis of T1DM.

5. The use of the animal model prepared by the method according to claim 1, characterized in that, Used to screen or identify substances that can prevent or treat T1DM.

6. A composition for preparing a T1DM non-human mammalian model by the preparation method according to claim 1, characterized in that, The composition comprises: M1) CD8 specifically targets GAD + T cells; and M2) streptozotocin; Specifically, the T cells that specifically target GAD specifically bind to the antigenic epitope peptide represented by AFLHATDLL (SEQ ID NO.1); The animal in question is a mouse.

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