Method for establishing neuromyelitis optica pedigree disease animal model

Abstract

The invention belongs to the technical field of biomedicine, and relates to the technical field of neurology, immunology and transgenosis in biomedicine, in particular to an adoptive metastasis model of mice with optic neuromyelitis pedigree diseases and a method for establishing the adoptive metastasis model of the mice with optic neuromyelitis pedigree diseases. The invention further discloses application of the adoptive metastasis model of the mouse with the neuromyelitis optica pedigree disease. According to the application, the Tfh cells are used for adoptively transferring mice to construct a new NMOSD mouse model, and a support basis is provided for exploring the effect of the AQP4 reactive Tfh cells in the NMOSD pathogenesis by evaluating the phenotype and pathology of the new NMOSD mouse model. The model can also become a tool for screening treatment targets for treating the neuromyelitis optica pedigree disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and relates to the fields of neurology, immunology and transgenic technology in biomedicine. More specifically, it relates to a mouse adoptive transfer model of neuromyelitis optica spectrum disorder and a method for establishing the mouse adoptive transfer model of neuromyelitis optica spectrum disorder, as well as the use of the mouse adoptive transfer model of neuromyelitis optica spectrum disorder. Background Art

[0002] According to the literature, neuromyelitis optica spectrum disorder (NMOSD) is a heterogeneous disease characterized by nerve inflammation and demyelination, which can cause motor, visual and other nerve dysfunctions. In up to 80% of cases, NMOSD is caused by pathogenic anti-aquaporin 4 (AQP4) antibodies. As a clear immune target in NMOSD, AQP4 provides a reasonable basis for constructing animal models to study its pathogenesis and screen therapeutic targets.

[0003] It is the consensus of those skilled in the art that an ideal animal model for studying the above-mentioned diseases should be able to mimic the pathological characteristics of human AQP4 antibody-positive NMOSD, mainly including massive death of astrocytes and manifested as the absence of AQP4 and glial fibrillary acidic protein (GFAP), secondary death of oligodendrocytes and neurons, accompanied by infiltration of macrophages / microglia, neutrophils, eosinophils, T cells, B cells and complement deposition. Actively immunized animal models are the most widely used and can better reflect the generation process and development process of the disease, while passively immunized animal models are mainly used to explore the pathogenesis of the disease. At present, the animal models of AQP4 antibody-positive NMOSD in research practice mainly include types such as injection of AQP4 antibody / complement on the basis of experimental autoimmune encephalomyelitis (EAE), direct injection of AQP4 antibody / complement, intracranial injection of cytokines, and adoptive transfer of AQP4-reactive Th17 cells. The above-mentioned animal models are all established by passive immunization, lacking active immunization models, and thus lacking preclinical animal experiments related to NMOSD.

[0004] Based on the existing situation that multiple studies in the prior art have confirmed the abnormal proportions and functions of peripheral blood follicular helper T (Tfh) cells and B cells in patients with AQP4 antibody-positive NMOSD, the inventors of the present application intend to use Tfh cell adoptive transfer mice to construct a new NMOSD mouse model, and provide a support basis for exploring the role of AQP4-reactive Tfh cells in the pathogenesis of NMOSD by evaluating its phenotype and pathology. Summary of the Invention

[0005] The object of the present invention is to provide a mouse adoptive transfer model for neuromyelitis optica spectrum disorder and a method for establishing the mouse adoptive transfer model for neuromyelitis optica spectrum disorder based on the basis and current situation of the prior art.

[0006] A further object of the present invention is to provide the use of the mouse adoptive transfer model for neuromyelitis optica spectrum disorder.

[0007] Based on the scientific experiments of the prior art, the present invention provides a mouse adoptive transfer model for neuromyelitis optica spectrum disorder. In the present invention, Tfh cell adoptive transfer mice are used to construct a new NMOSD mouse model, and provide a support basis for exploring the role of AQP4-reactive Tfh cells in the pathogenesis of NMOSD by evaluating its phenotype and pathology. This model can also become a tool for screening therapeutic targets for treating neuromyelitis optica spectrum disorder.

[0008] The present invention provides a method for establishing an anti-neuromyelitis optica spectrum disorder mouse adoptive transfer model, including the steps of:

[0009] 1) Providing experimental materials

[0010] (1) Donor mice: C57BL / 6 strain, AQP4 gene knockout type, female, 8 - 12 weeks old; Recipient mice: C57BL / 6 strain, wild type, female, 6 - 8 weeks old, body weight 18 - 20 g;

[0011] (2) Immunogenic polypeptide: Human-derived AQP4 peptide segment p135 - 153, with the sequence of LVTPPSVVGGLGVTMVHGN, and polypeptide purity > 95%.

[0012] 2) Mouse model establishment

[0013] (1) Weigh the AQP4 peptide segment, add 20 μl of DMSO to dissolve it, and then add D-PBS buffer for mixing;

[0014] (2) Weigh inactivated Mycobacterium tuberculosis and add it to CFA for mixing;

[0015] (3) Mix the above solutions and push and pull with a syringe to make an oil-in-water emulsion;

[0016] (4) Anesthetize the donor mice with isoflurane;

[0017] (5) Eleven days after modeling, sacrifice the donor mice for the next experiment.

[0018] 3) Isolation of mouse spleen and lymph node cells

[0019] (1) Decapitate the donor mice and immerse them in 75% ethanol;

[0020] (2) Remove the spleens and lymph nodes (cervical, brachial plexus, axillary, and inguinal) of the mice and place them in EP tubes containing RPMI 1640 medium on ice respectively;

[0021] (3) Place a sieve in a culture dish, add RPMI 1640 medium, grind the lymph nodes, aspirate the medium to wash the sieve, and transfer the lymph node cell suspension to a centrifuge tube;

[0022] (4) Place a sieve in a culture dish, add mouse lymphocyte separation solution, grind the spleen, aspirate the separation solution to wash the sieve, transfer the spleen cell suspension to a centrifuge tube, cover it with RPMI 1640 medium and keep the liquid level boundary distinct;

[0023] (5) Centrifuge the lymph node cell suspension at room temperature;

[0024] (6) Set the parameters and centrifuge the spleen cell suspension at room temperature;

[0025] (7) Aspirate the lymphocyte layer, add RPMI 1640 medium to resuspend, and centrifuge at room temperature;

[0026] (8) Discard the supernatant, resuspend with flow buffer and count.

[0027] 4) Tfh polarization and flow cytometry identification of mouse lymph node cells

[0028] (1) Resuspend with lymphocyte culture medium and transfer to a culture dish, add mouse IL-6 (final concentration 20 ng / ml), IL-21 (final concentration 20 ng / ml), anti-IFN-γ monoclonal antibody (final concentration 10 μg / ml), anti-IL-4 monoclonal antibody (final concentration 10 μg / ml), and anti-TGF-β monoclonal antibody (final concentration 20 μg / ml);

[0029] (2) Incubate in a 37°C, 5% CO2 cell culture incubator;

[0030] (3) After 3 days, take a small amount of cell suspension into a flow tube, add flow buffer, and centrifuge at room temperature;

[0031] (4) Discard the supernatant, resuspend with flow buffer, add ZombieAqua dead / live cell dye, mix well and incubate at 4°C in the dark;

[0032] (5) Add flow buffer and centrifuge at room temperature;

[0033] (6) Discard the supernatant, resuspend with flow buffer, add 3 μl each of anti-mouse CD3-FITC, CD4-BV421, CD45-APC-Cy7, CD11b-APC, CXCR5-PE-Cy7, ICOS-PE, and PD1-BV605, mix well and incubate in the dark at 4 °C;

[0034] (7) Add flow buffer and centrifuge at room temperature;

[0035] (8) Discard the supernatant, resuspend with flow buffer and load onto the flow cytometer.

[0036] 5) Sorting and flow cytometry identification of total B cells in mice

[0037] (1) Aspirate spleen cells into a flow tube and resuspend with flow buffer;

[0038] (2) Add rat serum;

[0039] (3) Add Cocktail, mix and incubate at room temperature;

[0040] (4) Vortex RapidSpheres, add RapidSpheres, mix and incubate at room temperature;

[0041] (5) Add flow buffer, pipette to mix, place the flow tube in a magnetic pole and incubate at room temperature;

[0042] (6) Remove the magnetic pole and invert the magnet and flow tube, pour the enriched cell suspension into a new flow tube;

[0043] (7) Take the cell suspension, add anti-mouse CD19-PE, mix well and incubate in the dark at 4 °C;

[0044] (8) Add flow buffer and centrifuge at room temperature;

[0045] (9) Discard the supernatant, resuspend with flow buffer and load onto the flow cytometer.

[0046] 6) Adoptive transfer of Tfh cells and total B cells

[0047] (1) Aspirate Tfh cells or total B cells into an EP tube and resuspend with flow buffer;

[0048] (2) Aspirate the above cell suspension with an insulin syringe and inject it into the recipient mice via the tail vein. The control group is injected with flow buffer via the tail vein;

[0049] (3) Inject PTX into the recipient mice via the intraperitoneal route on day 0 and day 2;

[0050] *The Tfh cell group was the experimental group, the B cell group was the isotype control, and the control group was the negative control.

[0051] 7) Phenotypic evaluation

[0052] (1) Behavioral evaluation: From the 5th day after adoptive transfer, the behavior of the mice was evaluated daily using the modified Kono's 5-point scoring system: 0 points: no disease; 0.5 points: partial weakness of the tail or slightly abnormal gait; 1.0 points: complete weakness of the tail or both partial weakness of the tail and slightly abnormal gait; 1.5 points: complete weakness of the tail and slightly weak hindlimbs; 2.0 points: complete weakness of the tail and moderately weak hindlimbs (evaluation criterion: the hindlimbs frequently fall down when climbing over the bars of the cage); 2.5 points: need to drag the hindlimbs to move; 3.0 points: complete paralysis of the hindlimbs; 3.5 points: complete paralysis of the hindlimbs and slightly weak forelimbs; 4.0 points: complete paralysis of all four limbs, only the head can move; 4.5 points: on the verge of death; 5.0 points: death;

[0053] (2) Body weight and change value: From the 5th day after adoptive transfer, the mice were weighed daily, and the change value compared to the initial body weight on the 0th day was calculated.

[0054] 8) Tissue sample preparation

[0055] (1) Mice at the peak of disease onset were taken and chloral hydrate was injected intraperitoneally;

[0056] (2) After anesthetizing the mice, they were laid on their backs, spread out and fixed on a mouse board, and the abdomen was disinfected with 75% ethanol;

[0057] (3) The chest walls on both sides were cut open along the lower edge of the costal arch with scissors, the chest walls on both sides were turned upwards to expose the heart, the pericardium was removed, and an appropriate amount of D-PBS buffer was dripped to keep it moist;

[0058] (4) A butterfly needle was inserted into the left cardiac apex, cold D-PBS buffer was perfused, and at the same time the right atrial appendage was cut open to drain the blood, and the color of the liver was observed to gradually turn white;

[0059] (5) Cold 4% paraformaldehyde was perfused, and when the twitching of the mice stopped, the tissues and organs became hardened;

[0060] (6) Decapitation was performed, and the brain, lumbar enlargement of the spinal cord, and the left optic nerve were quickly removed, and the above tissues were placed in 4% paraformaldehyde for fixation overnight;

[0061] (7) After washing with water, the above tissues were placed in a centrifuge tube;

[0062] (8) Dehydration: 70% alcohol for 1 hour → 85% alcohol for 1 hour → 90% alcohol for 1 hour → 95% alcohol for 0.5 hour → absolute ethanol (I) for 20 minutes → absolute ethanol (II) for 20 minutes;

[0063] (9) Transparency: Anhydrous ethanol + xylene (1:1) for 10 minutes → Xylene (I) for 10 minutes → Xylene (II) for 7 minutes;

[0064] (10) Wax infiltration and embedding: Paraffin (I) (60°C) for 1 hour → Paraffin (II) (60°C) for 1 hour → Paraffin (III) (60°C) for 1 hour;

[0065] (11) Sectioning: Sequentially cut out the striatal plane of the brain, the plane of the third ventricle / hippocampus of the brain, the transverse section of the lumbar enlargement of the spinal cord, and the longitudinal section of the optic nerve according to the atlas;

[0066] (12) Mounting: Immerse the sections in a warm water bath, let the sections flatten naturally, insert the adhesive-free glass slides obliquely into the water surface to pick up the sections, so that the sections adhere to the glass slides, and place them in an oven for baking for later use.

[0067] 9) Histological HE staining

[0068] (1) Take out the sections, dewax to water: Xylene (I) for 5 minutes → Xylene (II) for 5 minutes → Anhydrous ethanol (I) for 5 minutes → Anhydrous ethanol (II) for 5 minutes → 95% ethanol for 5 minutes → 85% ethanol for 5 minutes → 75% ethanol for 5 minutes → 50% ethanol for 5 minutes → Pure water for 5 minutes;

[0069] (2) Stain with hematoxylin staining solution at room temperature and rinse with tap water;

[0070] (3) Differentiate with differentiating solution and rinse with tap water;

[0071] (4) Stain with eosin staining solution at room temperature and rinse with tap water;

[0072] (5) Dehydration and transparency: 95% ethanol (I) for 2 - 3 seconds → 95% ethanol (II) for 2 - 3 seconds → Anhydrous ethanol for 5 minutes → Anhydrous ethanol + xylene (1:1) for 5 minutes → Xylene (I) for 2 minutes → Xylene (II) for 2 minutes;

[0073] (6) Mount the sections with neutral balsam, observe under the microscope and take pictures.

[0074] 10) Histological LFB myelin staining

[0075] (1) Take out the sections, dewax to water: Xylene (I) for 5 minutes → Xylene (II) for 5 minutes → Anhydrous ethanol (I) for 5 minutes → Anhydrous ethanol (II) for 5 minutes → 95% ethanol for 5 minutes → 85% ethanol for 5 minutes → 75% ethanol for 5 minutes → 50% ethanol for 5 minutes → Pure water for 5 minutes;

[0076] (2) Incubate with Luxol fastblue staining solution overnight at room temperature;

[0077] (3) Wash away the excess staining solution with 95% ethanol and rinse with tap water;

[0078] (4)Differentiate with Luxol differentiating solution;

[0079] (5)Differentiate with 70% ethanol, rinse with tap water, and observe under the microscope until the contours of gray and white matter are distinct;

[0080] (6)Counterstain with cresyl violet staining solution at room temperature, and rinse with tap water;

[0081] (7)Dehydrate and clear: 95% ethanol (I) for 2 - 3 seconds → 95% ethanol (II) for 2 - 3 seconds → absolute ethanol for 5 minutes → absolute ethanol + xylene (1:1) for 5 minutes → xylene (I) for 2 minutes → xylene (II) for 2 minutes;

[0082] (8)Mount the section with neutral balsam, observe under the microscope and take pictures.

[0083] 11) Immunohistochemistry

[0084] (1)Take out the section, dewax to water: xylene (I) for 5 minutes → xylene (II) for 5 minutes → absolute ethanol (I) for 5 minutes → absolute ethanol (II) for 5 minutes → 95% ethanol for 5 minutes → 85% ethanol for 5 minutes → 75% ethanol for 5 minutes → 50% ethanol for 5 minutes → pure water for 5 minutes;

[0085] (2)Boil the sodium citrate / EDTA antigen retrieval solution in a microwave oven → put in the section → high heat for 5 minutes, cool, repeat 2 - 3 times, and cool naturally;

[0086] (3)Add 3% hydrogen peroxide solution and incubate the section at room temperature;

[0087] (4)Wash with D-PBS buffer;

[0088] (5)Add 10% BSA and block at room temperature in a humidified chamber;

[0089] (6)Discard the blocking solution, draw a hydrophobic circle at the edge of the sample and dry it;

[0090] (7)Dilute the primary antibody at 1:1000 and add it to the section, drop the antibody on each tissue, and incubate overnight at 4°C in a humidified chamber;

[0091] (8)Wash with D-PBS buffer;

[0092] (9)Dilute the secondary antibody at 1:1000 and add it to the section, drop the antibody on each tissue, and incubate at room temperature in a humidified chamber: (10)Wash with D-PBS buffer;

[0093] (11)Prepare the AB mixture, drop 50 μl on each tissue, and incubate at room temperature in a humidified chamber;

[0094] (12)Wash with D-PBS buffer;

[0095] (13) Prepare DAB staining solution, add 50 μl to each tissue, control the color development under the microscope, and rinse with tap water: (14) Stain with hematoxylin staining solution at room temperature and rinse with tap water:

[0096] (15) Dehydration and clearing: 95% ethanol (I) for 2 - 3 seconds → 95% ethanol (II) for 2 - 3 seconds → absolute ethanol for 5 minutes → absolute ethanol + xylene (1:1) for 5 minutes → xylene (I) for 2 minutes → xylene (II) for 2 minutes. (16) Mount the slides with neutral balsam, observe under the microscope and take pictures.

[0097] The present invention further carried out flow cytometry identification, phenotypic evaluation, histological staining and immunohistochemistry experiments: The experimental results showed that the adoptive transfer model of Tfh cells in mice with neuromyelitis optica spectrum disorder established by the present invention confirmed that AQP4 - reactive Tfh cells had certain pathogenicity. In this model, astrocytes and microglia were significantly activated, chemotaxis was enhanced, there was evidence of demyelination, and a large number of activated microglia / macrophages and lymphocytes infiltrated in the central nervous system. However, the adoptive transfer of B cells could not form a model, which confirmed that B cells themselves were difficult to break through the blood - brain barrier and enter the central nervous system to cause disease, and needed to rely on the driving effect of T cells.

[0098] The present invention successfully constructed an adoptive transfer model of mice with neuromyelitis optica spectrum disorder, which provided a support basis for exploring the role of AQP4 - reactive Tfh cells in the pathogenesis of NMOSD by evaluating its phenotype and pathology. This model can also become a tool for screening therapeutic targets for neuromyelitis optica spectrum disorder. Description of the Drawings

[0099] Figure 1 Flow cytometry identification of Tfh cells. Among them, A: Circulate live cells from mouse lymph node cells; B: Remove adherent bodies; C: Circulate total lymphocytes; D: Circulate CD3 + CD4 + T cells; E: Identify the positive proportion of CXCR5. The red color is the addition of CXCR5 - PE - Cy7 flow antibody, and the blue color is the isotype control; F: Identify the positive proportion of ICOS. The red color is the addition of ICOS - PE flow antibody, and the blue color is the isotype control; G: Identify the positive proportion of PD1. The red color is the addition of PD1 - BV421 flow antibody, and the blue color is the isotype control.

[0100] Figure 2 Flow cytometry identification of B cells. Among them, A: Remove adherent bodies; B: Circulate total lymphocytes; C: Identify the positive proportion of CD19. The red color is the addition of CD19 - PE flow antibody, and the blue color is the isotype control.

[0101] Figure 3 Phenotypic evaluation of mice in each group (N = 6). Among them, A: Behavioral score; B: Body weight; C: Value of body weight change.

[0102] Figure 4 For each group, HE staining of the optic nerves was performed. Among them, A: adoptive transfer group of Tfh cells; B: adoptive transfer group of B cells; C: control group (scale bar is 100 μm).

[0103] Figure 5 For each group, HE staining of the spinal cords was performed. Among them, A: adoptive transfer group of Tfh cells; B: adoptive transfer group of B cells; C: control group (scale bar is 100 μm).

[0104] Figure 6 For each group, HE staining of the brains was performed. Among them, A: adoptive transfer group of Tfh cells; B: adoptive transfer group of B cells; C: control group (scale bar is 500 μm).

[0105] Figure 7 For each group, LFB myelin staining of the optic nerves was performed. Among them, A: adoptive transfer group of Tfh cells; B: adoptive transfer group of B cells; C: control group (scale bar is 100 μm).

[0106] Figure 8 For each group, LFB myelin staining of the spinal cords was performed. Among them, A: adoptive transfer group of Tfh cells; B: adoptive transfer group of B cells; C: control group (scale bar is 100 μm).

[0107] Figure 9 For each group, LFB myelin staining of the brains was performed. Among them, A: adoptive transfer group of Tfh cells; B: adoptive transfer group of B cells; C: control group (scale bar is 500 μm).

[0108] Figure 10 The proportions of inflammatory cell infiltration and demyelination in each group (N = 3). *P < 0.05; **P < 0.01; ***P < 0.001. Among them, A: proportion of inflammatory cell infiltration in the optic nerves; B: proportion of inflammatory cell infiltration in the spinal cords; C: proportion of inflammatory cell infiltration in the brains; D: proportion of demyelination in the optic nerves; E: proportion of demyelination in the spinal cords; F: proportion of demyelination in the brains.

[0109] Figure 11 For each group, GFAP immunohistochemistry of the spinal cords was performed. Among them, A: adoptive transfer group of Tfh cells; B: adoptive transfer group of B cells; C: control group (scale bar is 100 μm).

[0110] Figure 12 For each group, AQP4 immunohistochemistry of the spinal cords was performed. A: adoptive transfer group of Tfh cells; B: adoptive transfer group of B cells; C: control group (scale bar is 100 μm).

[0111] Figure 13 For each group, MBP immunohistochemistry of the spinal cords was performed. Among them, A: adoptive transfer group of Tfh cells; B: adoptive transfer group of B cells; C: control group (scale bar is 100 μm).

[0112] Figure 14 Immunohistochemistry of Iba1 in each group of spinal cords. Among them, A: adoptive transfer group of Tfh cells; B: adoptive transfer group of B cells; C: control group (scale bar: 100 μm).

[0113] Figure 15 Immunohistochemistry of CD3 in each group of spinal cords. Among them, A: adoptive transfer group of Tfh cells; B: adoptive transfer group of B cells; C: control group (scale bar: 100 μm).

[0114] Figure 16 Immunohistochemistry of B220 in each group of spinal cords. Among them, A: adoptive transfer group of Tfh cells; B: adoptive transfer group of B cells; C: control group (scale bar: 100 μm).

[0115] Figure 17 Immunohistochemistry of Ly6g in each group of spinal cords. Among them, A: adoptive transfer group of Tfh cells; B: adoptive transfer group of B cells; C: control group (scale bar: 100 μm).

[0116] Figure 18 Immunohistochemistry of CXCL13 in each group of spinal cords. Among them, A: adoptive transfer group of Tfh cells; B: adoptive transfer group of B cells; C: control group (scale bar: 100 μm).

[0117] Figure 19 Immunohistochemistry was used to detect the protein expression levels in the spinal cords of each group (N = 3), *P < 0.05; **P < 0.01; ***P < 0.001. Among them, A: average optical density value of GFAP; B: average optical density value of AQP4; C: average optical density value of MBP; D: average optical density value of Iba1; E: average optical density value of CD3; F: average optical density value of B220; G: average optical density value of Ly6g; H: average optical density value of CXCL13. Detailed implementation manners

[0118] Example 1

[0119] Through the following methods and steps: establishing an adoptive transfer model of mice with neuromyelitis optica spectrum disorder

[0120] 1) Provide experimental materials

[0121] (1) Donor mice: C57BL / 6 strain, AQP4 gene knockout type, female, 8 - 12 weeks old, purchased from Cyagen Biosciences (Suzhou) Inc.; Recipient mice: C57BL / 6 strain, wild type, female, 6 - 8 weeks old, weighing 18 - 20 g, purchased from Shanghai JieSaiJie Laboratory Animal Co., Ltd.

[0122] (2) Immunogenic polypeptide: The human-derived AQP4 peptide segment p135-153 with the sequence LVTPPSVVGGLGVTMVHGN was synthesized by Suzhou Qiangyao Biotechnology Co., Ltd., and the polypeptide purity > 95%.

[0123] 2) Mouse model establishment

[0124] (1) Weigh 1 mg of AQP4 peptide segment, add 20 μl of DMSO to dissolve it, and then add 980 μl of D-PBS buffer to mix;

[0125] (2) Weigh 3 mg of inactivated Mycobacterium tuberculosis and add 1 ml of CFA to mix;

[0126] (3) Mix the above solutions in a 1:1 ratio, and repeatedly push and pull with a syringe on ice for 1 hour to prepare an oil-in-water emulsion;

[0127] (4) Anesthetize the donor mice with isoflurane and subcutaneously inject the emulsion at 4 points on the back of the mice, 50 μl per point, 200 μl per mouse;

[0128] (5) Eleven days after model establishment, sacrifice the donor mice for the next experiment.

[0129] 3) Isolation of mouse spleen and lymph node cells

[0130] (1) Decapitate the donor mice and immerse them in 75% ethanol;

[0131] (2) Take out the mouse spleen and lymph nodes (neck, brachial plexus, axilla, and groin) in a laminar flow hood and place them in EP tubes containing RPMI 1640 medium on ice respectively;

[0132] (3) Place a 70 μm sieve in a 60 mm culture dish, add 5 ml of RPMI 1640 medium, gently grind the lymph nodes with the inner core of a 5 ml syringe, continuously suck the medium to wash the sieve, and transfer the lymph node cell suspension to a 15 ml centrifuge tube;

[0133] (4) Place a 70 μm sieve in a 60 mm culture dish, add 5 ml of mouse lymphocyte separation solution, gently grind the spleen with the inner core of a 5 ml syringe, continuously suck the separation solution to wash the sieve, transfer the spleen cell suspension to a 15 ml centrifuge tube, and cover it with 1 ml of RPMI1640 medium to keep the liquid interface distinct;

[0134] (5) Centrifuge the lymph node cell suspension at 1000 rpm at room temperature for 5 minutes;

[0135] (6) Set the parameters to centrifuge the spleen cell suspension at 3,2,800 g at room temperature for 20 minutes;

[0136] (7) Aspirate the lymphocyte layer, add 5 ml of RPMI 1640 medium to resuspend, and centrifuge at 1000 rpm for 5 minutes at room temperature;

[0137] (8) Discard the supernatant, resuspend with an appropriate amount of flow buffer and count.

[0138] 4) Tfh polarization and flow cytometry identification of mouse lymph node cells

[0139] (1) Add 5 ml of lymphocyte culture medium to resuspend and transfer to a 60 mm culture dish. Add mouse IL-6 (final concentration 20 ng / ml), IL-21 (final concentration 20 ng / ml), anti-IFN-γ monoclonal antibody (final concentration 10 μg / ml), anti-IL-4 monoclonal antibody (final concentration 10 μg / ml), and anti-TGF-β monoclonal antibody (final concentration 20 μg / ml);

[0140] (2) Incubate in a 37°C, 5% CO2 cell culture incubator for 3 days;

[0141] (3) After 3 days, take a small amount of cell suspension into a 5 ml flow tube, add 1 ml of flow buffer, and centrifuge at 1000 rpm for 5 minutes at room temperature;

[0142] (4) Discard the supernatant, add 100 μl of flow buffer to resuspend, add 0.5 μl of ZombieAqua dead / live cell dye, mix well and incubate at 4°C in the dark for 30 minutes;

[0143] (5) Add 1 ml of flow buffer, centrifuge at 1000 rpm for 5 minutes at room temperature;

[0144] (6) Discard the supernatant, add 100 μl of flow buffer to resuspend, add 3 μl each of anti-mouse CD3-FITC, CD4-BV421, CD45-APC-Cy7, CD11b-APC, CXCR5-PE-Cy7, ICOS-PE, and PD1-BV605, mix well and incubate at 4°C in the dark for 30 minutes;

[0145] (7) Add 1 ml of flow buffer, centrifuge at 1000 rpm for 5 minutes at room temperature;

[0146] (8) Discard the supernatant, add 250 μl of flow buffer to resuspend and load onto the machine.

[0147] 5) Sorting and flow cytometry identification of mouse total B cells

[0148] (1) Aspirate approximately 1x10 8 spleen cells into a 5 ml flow tube, add 1 ml of flow buffer to resuspend;

[0149] (2) Add 50 μl of rat serum;

[0150] (3) Add 50 μl of Cocktail, mix and incubate at room temperature for 5 minutes;

[0151] (4) Vortex RapidSpheres for 30 seconds, add 50 μL of RapidSpheres, mix and incubate at room temperature for 2.5 minutes;

[0152] (5) Add flow buffer to 2.5 ml, pipette up and down several times to mix, place the flow tube in the magnetic pole and incubate at room temperature for 2.5 minutes;

[0153] (6) Pick up the magnetic pole and invert the magnet and the flow tube, pour the enriched cell suspension into a new flow tube;

[0154] (7) Take a small amount of cell suspension, add 5 μl of anti-mouse CD19-PE, mix well and incubate at 4 °C in the dark for 30 minutes; (8) Add 1 ml of flow buffer, centrifuge at 1000 rpm at room temperature for 5 minutes;

[0155] (9) Discard the supernatant, add 250 μl of flow buffer to resuspend and load onto the machine.

[0156] 6) Adoptive transfer of Tfh cells and total B cells

[0157] (1) Pipette approximately 2×10 7 Tfh cells or total B cells into an EP tube, add flow buffer to 0.2 ml to resuspend;

[0158] (2) Pipette the above cell suspension with a 1 ml insulin syringe and inject it into the recipient mice via the tail vein at a dose of 0.2 ml per mouse. The control group is injected with 0.2 ml of flow buffer per mouse via the tail vein;

[0159] (3) Inject PTX into the recipient mice via the intraperitoneal route on day 0 and day 2 at a dose of 200 ng per mouse per injection;

[0160] *The Tfh cell group is the experimental group, the B cell group is the isotype control, and the control group is the negative control.

[0161] 7) Phenotypic evaluation

[0162] (1) Behavioral assessment: Starting from the 5th day after adoptive transfer, the behavior of the mice was evaluated daily. The modified Kono's 5-point evaluation criteria were used: 0 points: no disease; 0.5 points: partial weakness of the tail or slightly abnormal gait; 1.0 points: complete weakness of the tail or both partial weakness of the tail and slightly abnormal gait; 1.5 points: complete weakness of the tail and slightly weak hind limbs; 2.0 points: complete weakness of the tail and moderately weak hind limbs (evaluation criteria: when climbing over the bars of the cage, the hind limbs frequently drop down); 2.5 points: need to drag the hind limbs to move; 3.0 points: complete paralysis of the hind limbs; 3.5 points: complete paralysis of the hind limbs and slightly weak forelimbs; 4.0 points: complete paralysis of all four limbs, only the head can move; 4.5 points: on the verge of death; 5.0 points: death;

[0163] (2) Body weight and change value: Starting from the 5th day after adoptive transfer, the mice were weighed daily, and the change value compared to the initial body weight on the 0th day was calculated.

[0164] 8) Preparation of tissue samples

[0165] (1) Select mice at the peak of disease onset, and intraperitoneally inject 0.2 ml of 4% chloral hydrate;

[0166] (2) After anesthetizing the mice, place them in a supine position, spread and fix them on a mouse board, and disinfect the abdomen with 75% ethanol;

[0167] (3) Use scissors to cut open both sides of the chest along the lower edge of the costal arch, turn up both sides of the chest to expose the heart, remove the pericardium with forceps, and drip an appropriate amount of D-PBS buffer to keep it moist;

[0168] (4) Insert a butterfly needle into the apex of the left heart, perfuse 20 ml of cold D-PBS buffer, and at the same time cut open the right atrial appendage to drain the blood, and observe the liver color gradually turning white;

[0169] (5) Perfuse 20 ml of cold 4% paraformaldehyde, wait until the mice stop twitching completely, and the whole body tissues and organs become hard;

[0170] (6) Decapitate, quickly remove the brain, lumbar enlargement of the spinal cord and the left optic nerve, and place the above tissues in 4% paraformaldehyde at 4°C for fixation overnight;

[0171] (7) After washing with water, place the above tissues in a 15 ml centrifuge tube;

[0172] (8) Dehydration: 70% alcohol for 1 hour → 85% alcohol for 1 hour → 90% alcohol for 1 hour → 95% alcohol for 0.5 hour → absolute ethanol (I) for 20 minutes → absolute ethanol (II) for 20 minutes;

[0173] (9) Clearing: absolute ethanol + xylene (1:1) for 10 minutes → xylene (I) for 10 minutes → xylene (II) for 7 minutes;

[0174] (10) Paraffin infiltration and embedding: Paraffin (I) (60°C) for 1 hour → Paraffin (II) (60°C) for 1 hour → Paraffin (III) (60°C) for 1 hour;

[0175] (11) Sectioning: Sequentially cut out the striatal plane of the brain, the plane of the third ventricle / hippocampus of the brain, the transverse section of the lumbar enlargement of the spinal cord, and the longitudinal section of the optic nerve according to the atlas;

[0176] (12) Mounting: Immerse the sections in a warm water bath. The sections will flatten naturally under the action of surface tension. Insert the adhesive-free glass slide obliquely into the water surface to pick up the sections, so that the sections adhere to the glass slide, and place them in an oven at 60°C for 2 hours for baking and then reserve.

[0177] 9) Histological HE staining

[0178] (1) Take out the sections and dewax to water: Xylene (I) for 5 minutes → Xylene (II) for 5 minutes → Absolute ethanol (I) for 5 minutes → Absolute ethanol (II) for 5 minutes → 95% ethanol for 5 minutes → 85% ethanol for 5 minutes → 75% ethanol for 5 minutes → 50% ethanol for 5 minutes → Pure water for 5 minutes;

[0179] (2) Stain with hematoxylin staining solution at room temperature for 5 minutes, and rinse with tap water for 10 minutes;

[0180] (3) Differentiate with differentiating solution for 30 seconds, and rinse with tap water for 10 minutes;

[0181] (4) Stain with eosin staining solution at room temperature for 2 minutes, and rinse with tap water for 10 minutes;

[0182] (5) Dehydration and clearing: 95% ethanol (I) for 2 - 3 seconds → 95% ethanol (II) for 2 - 3 seconds → Absolute ethanol for 5 minutes → Absolute ethanol + xylene (1:1) for 5 minutes → Xylene (I) for 2 minutes → Xylene (II) for 2 minutes;

[0183] (6) Mount the sections with neutral balsam, observe under the microscope and take pictures.

[0184] 10) Histological LFB myelin staining

[0185] (1) Take out the sections and dewax to water: Xylene (I) for 5 minutes → Xylene (II) for 5 minutes → Absolute ethanol (I) for 5 minutes → Absolute ethanol (II) for 5 minutes → 95% ethanol for 5 minutes → 85% ethanol for 5 minutes → 75% ethanol for 5 minutes → 50% ethanol for 5 minutes → Pure water for 5 minutes;

[0186] (2) Stain with Luxol fast blue staining solution overnight at room temperature;

[0187] (3) Wash off the excess staining solution with 95% ethanol, and rinse with tap water for 10 minutes;

[0188] (4) Differentiate with Luxol differentiating solution for 15 seconds;

[0189] (5) Differentiate with 70% ethanol for 30 seconds, rinse with tap water for 10 minutes, and observe under the microscope until the outlines of gray and white matter are distinct;

[0190] (6) Counterstain with tar purple staining solution at room temperature for 30 seconds, and rinse with tap water for 10 minutes;

[0191] (7) Dehydration and clearing: 95% ethanol (I) for 2 - 3 seconds → 95% ethanol (II) for 2 - 3 seconds → absolute ethanol for 5 minutes → absolute ethanol + xylene (1:1) for 5 minutes → xylene (I) for 2 minutes → xylene (II) for 2 minutes;

[0192] (8) Mount the section with neutral balsam, observe under the microscope and take pictures.

[0193] 11) Immunohistochemistry

[0194] (1) Take out the section, dewax to water: xylene (I) for 5 minutes → xylene (II) for 5 minutes → absolute ethanol (I) for 5 minutes → absolute ethanol (II) for 5 minutes → 95% ethanol for 5 minutes → 85% ethanol for 5 minutes → 75% ethanol for 5 minutes → 50% ethanol for 5 minutes → pure water for 5 minutes;

[0195] (2) Boil the sodium citrate / EDTA antigen retrieval solution in a microwave oven → put in the section → high fire for 5 minutes, cool for 10 minutes, repeat 2 - 3 times, and cool naturally;

[0196] (3) Add 3% hydrogen peroxide solution and incubate the section at room temperature for 10 minutes;

[0197] (4) Wash with D - PBS buffer 3 times, 5 minutes each time;

[0198] (5) Add 10% BSA and block at room temperature in a humidified chamber for 1 hour;

[0199] (6) Discard the blocking solution, draw a hydrophobic circle 2 - 3 mm away from the edge of the sample and dry for 1 minute;

[0200] (7) Dilute the primary antibody at 1:1000 and add it to the section, drop 50 μl of antibody on each tissue, and incubate at 4°C overnight in a humidified chamber;

[0201] (8) Wash with D - PBS buffer 3 times, 5 minutes each time;

[0202] (9) Dilute the secondary antibody at 1:1000 and add it to the section, drop 50 μl of antibody on each tissue, and incubate at room temperature for 30 minutes in a humidified chamber;

[0203] (10) Wash with D - PBS buffer 3 times, 5 minutes each time;

[0204] (11) Prepare the AB mixture and add 50 μl to each tissue, incubate at room temperature in a wet box for 3 hours:

[0205] (12) Wash with D-PBS buffer 3 times, 5 minutes each time:

[0206] (13) Prepare the DAB staining solution, add 50 μl to each tissue, control the color development under the microscope, and rinse with tap water for 10 minutes:

[0207] (14) Stain with hematoxylin staining solution at room temperature for 1 - 2 minutes, and rinse with tap water for 10 minutes:

[0208] (15) Dehydration and clearing: 95% ethanol (I) for 2 - 3 seconds → 95% ethanol (II) for 2 - 3 seconds → absolute ethanol for 5 minutes → absolute ethanol + xylene (1:1) for 5 minutes → xylene (I) for 2 minutes → xylene (II) for 2 minutes. (16) Mount with neutral balsam, observe under the microscope and take pictures.

[0209] Example 2 Experimental Detection

[0210] 1. Flow cytometry identification

[0211] (1) Flow cytometry identification of Tfh cells: After 3 days of Tfh polarization of mouse lymph node cells, the positive ratio of CXCR5 was 64.4%, the positive ratio of ICOS was 80.8%, and the positive ratio of PD1 was 44.7% (see Figure 1 ).

[0212] (2) Flow cytometry identification of B cells: After magnetic bead negative sorting, the proportion of CD19 + B cells in mouse spleen cells was 91.8% (see Figure 2 ).

[0213] 2. Phenotypic evaluation

[0214] (1) Behavioral scoring: Starting from the 6th day after adoptive transfer, the mice in the Tfh cell adoptive transfer group began to show phenotypes and gradually worsened. First, it was manifested as partial weakness of the tail, reaching the peak on the 10th day, manifested as complete weakness of the tail and moderate weakness of the hind limbs, with an average score of 2.0 ± 0.3 points, and then gradually relieved, with an average score of 0.2 ± 0.3 points on the 21st day. The mice in the B cell adoptive transfer group and the control group did not show phenotypes within 21 days after adoptive transfer, and the average scores were both 0 points (see Figure 3 A).

[0215] (2) Body weight: Compared with the other two groups of mice, the mice in the adoptive transfer group of Tfh cells showed a significant decrease in body weight on the 5th day after adoptive transfer, and reached the lowest value on the 9th day, with an average body weight of 16.89 ± 1.17 g. Then the body weight gradually increased, and the average body weight on the 21st day was 21.63 ± 0.67 g. The body weights of the mice in the adoptive transfer group of B cells and the control group of mice did not show a significant decrease. The average body weights on the 21st day were 23.01 ± 0.96 g and 22.92 ± 0.61 g respectively (see Figure 3 B).

[0216] (3) Body weight change value: The initial body weight of the mice in the adoptive transfer group of Tfh cells was 19.16 ± 0.58 g, the initial body weight of the mice in the adoptive transfer group of B cells was 19.02 ± 0.63 g, and the initial body weight of the mice in the adoptive transfer group of the control group was 19.10 ± 0.47 g. There was no significant difference among the groups (P = 0.911). Compared with the initial body weight, the body weight of the mice in the adoptive transfer group of Tfh cells decreased on the 5th day after adoptive transfer and reached the maximum change value of -2.27 ± 0.90 g on the 9th day. Then the body weight change value decreased to 0 and gradually increased, and the body weight change value on the 21st day was 2.47 ± 0.54 g. The body weight change values of the mice in the adoptive transfer group of B cells and the control group of mice gradually increased, and the body weight change values on the 21st day were 3.99 ± 0.43 g and 3.81 ± 0.32 g respectively (see Figure 3 C).

[0217] 3. Histological staining

[0218] (1) HE staining of the optic nerve: The HE staining of the optic nerve in each group is as Figure 4 shown. The proportions of inflammatory cell infiltration in the optic nerves of the adoptive transfer group of Tfh cells, the adoptive transfer group of B cells, and the control group were 2.20 ± 0.43%, 0.24 ± 0.06%, and 0.24 ± 0.09% respectively. There was a highly significant difference among the groups (P = 0.008) (see Figure 10 A). (2) HE staining of the spinal cord: The HE staining of the spinal cord in each group is as Figure 5 shown. The proportions of inflammatory cell infiltration in the spinal cords of the adoptive transfer group of Tfh cells, the adoptive transfer group of B cells, and the control group were 3.17 ± 0.38%, 0.05 ± 0.05%, and 0.05 ± 0.05% respectively. There was a highly significant difference among the groups (P = 0.001) (see Figure 10 B).

[0219] (3) HE staining of the brain: The HE staining of the brain in each group is as Figure 6As shown. The proportions of cerebral inflammatory cell infiltration in the adoptive transfer group of Tfh cells, the adoptive transfer group of B cells, and the control group were 0.20±0.06%, 0.03±0.01%, and 0.03±0.02%, respectively, and the differences among the groups were highly statistically significant (P = 0.001) (see Figure 10 C).

[0220] (4) LFB myelin staining of the optic nerve: The LFB myelin staining of the optic nerve in each group was as Figure 7 shown. The proportions of demyelination of the optic nerve in the adoptive transfer group of Tfh cells, the adoptive transfer group of B cells, and the control group were 2.10±0.35%, 0.17±0.04%, and 0.22±0.11%, respectively, and the differences among the groups were highly statistically significant (P<0.001) (see Figure 10 D).

[0221] (5) LFB myelin staining of the spinal cord: The LFB myelin staining of the spinal cord in each group was as Figure 8 shown. The proportions of demyelination of the spinal cord in the adoptive transfer group of Tfh cells, the adoptive transfer group of B cells, and the control group were 1.16±0.24%, 0.07±0.01%, and 0.08±0.02%, respectively, and the differences among the groups were highly statistically significant (P<0.001) (see Figure 10 E). (6) LFB myelin staining of the brain: The LFB myelin staining of the brain in each group was as Figure 9 shown. The proportions of demyelination of the brain in the adoptive transfer group of Tfh cells, the adoptive transfer group of B cells, and the control group were 4.27±0.39%, 1.31±0.58%, and 1.08±0.49%, respectively, and the differences among the groups were highly statistically significant (P<0.001) (see Figure 10 F).

[0222] 4. Immunohistochemistry:

[0223] (1) GFAP: The GFAP immunohistochemistry of the spinal cord in each group was as Figure 11 shown. The average optical density values of GFAP in the adoptive transfer group of Tfh cells, the adoptive transfer group of B cells, and the control group were 0.286±0.002, 0.191±0.009, and 0.205±0.018, respectively, and the differences among the groups were highly statistically significant (P<0.001) (see Figure 19 A). (2) AQP4: The AQP4 immunohistochemistry of the spinal cord in each group was as Figure 12 shown. The average optical density values of AQP4 in the adoptive transfer group of Tfh cells, the adoptive transfer group of B cells, and the control group were 0.274±0.009, 0.276±0.022, and 0.269±0.023, respectively, and the differences among the groups were not statistically significant (P = 0.891) (see Figure 19 B).

[0224] (3) MBP: The MBP immunohistochemistry of each group of spinal cords was as follows Figure 13 shown. The average optical density values of MBP in the spinal cords of the adoptive transfer group of Tfh cells, the adoptive transfer group of B cells, and the control group were 0.263 ± 0.002, 0.304 ± 0.022, and 0.310 ± 0.007, respectively. The differences among the groups were highly statistically significant (P = 0.009) (see Figure 19 C). (4) Iba1: The Iba1 immunohistochemistry of each group of spinal cords was as follows Figure 14 shown. The average optical density values of Iba1 in the spinal cords of the adoptive transfer group of Tfh cells, the adoptive transfer group of B cells, and the control group were 0.277 ± 0.013, 0.147 ± 0.034, and 0.139 ± 0.015, respectively. The differences among the groups were highly statistically significant (P < 0.001) (see Figure 19 D).

[0225] (5) CD3: The CD3 immunohistochemistry of each group of spinal cords was as follows Figure 15 shown. The average optical density values of CD3 in the spinal cords of the adoptive transfer group of Tfh cells, the adoptive transfer group of B cells, and the control group were 0.158 ± 0.006, 0.123 ± 0.009, and 0.124 ± 0.008, respectively. The differences among the groups were highly statistically significant (P = 0.002) (see Figure 19 E).

[0226] (6) B220: The B220 immunohistochemistry of each group of spinal cords was as follows Figure 16 shown. The average optical density values of B220 in the spinal cords of the adoptive transfer group of Tfh cells, the adoptive transfer group of B cells, and the control group were 0.174 ± 0.002, 0.156 ± 0.005, and 0.151 ± 0.006, respectively. The differences among the groups were highly statistically significant (P = 0.002) (see Figure 19 F). (7) Ly6g: The Ly6g immunohistochemistry of each group of spinal cords was as follows Figure 17 shown. The average optical density values of Ly6g in the spinal cords of the adoptive transfer group of Tfh cells, the adoptive transfer group of B cells, and the control group were 0.121 ± 0.003, 0.118 ± 0.012, and 0.121 ± 0.003, respectively. The differences among the groups were not statistically significant (P = 0.907) (see Figure 19 G).

[0227] (8) CXCL13: The CXCL13 immunohistochemistry of each group of spinal cords was as follows Figure 18 shown. The average optical density values of CXCL13 in the spinal cords of the adoptive transfer group of Tfh cells, the adoptive transfer group of B cells, and the control group were 0.160 ± 0.018, 0.112 ± 0.003, and 0.104 ± 0.007, respectively. The differences among the groups were highly statistically significant (P = 0.002) (see Figure 19 H).

[0228] The experimental results show that by successfully establishing an adoptive transfer model of Tfh cells in mice with neuromyelitis optica spectrum disorder (NMOSD), it is confirmed that AQP4-reactive Tfh cells have certain pathogenicity. In this model, astrocytes and microglia are significantly activated, chemotaxis is enhanced, there is evidence of demyelination, and there are a large number of activated microglia / macrophages and lymphocytes infiltrating the central nervous system. However, adoptive transfer of B cells fails to establish a model, confirming that B cells themselves are difficult to break through the blood-brain barrier and enter the central nervous system to cause disease, and they need to rely on the driving effect of T cells. The present invention constructs an adoptive transfer model of mice with NMOSD, which can provide a support basis for exploring the role of AQP4-reactive Tfh cells in the pathogenesis of NMOSD by evaluating its phenotype and pathology. This model can also become a tool for screening therapeutic targets for treating NMOSD.

Claims

1. A method for establishing an animal model of neuromyelitis optica spectrum disorder, characterized in that, Including the following steps: 1) Provide experimental materials, including donor mice, recipient mice, and immunogenic polypeptides; 2) Establish a mouse model; 3) Isolate spleen and lymph node cells from mice; 4) Polarize and identify Tfh cells in mouse lymph node cells by flow cytometry; 5) Sort and identify total B cells in mice by flow cytometry; 6) Adoptively transfer Tfh cells and total B cells; 7) Perform phenotypic evaluation; 8) Prepare tissue samples; 9) Perform histological HE staining; 10) Perform histological LFB myelin staining; 11) Perform immunohistochemistry.

2. The method according to claim 1, characterized in that, The step 1) of providing experimental materials includes: (1) Donor mice: C57BL / 6 strain, AQP4 gene knockout type, female, 8 - 12 weeks old; Recipient mice: C57BL / 6 strain, wild type, female, 6 - 8 weeks old, weighing 18 - 20 g; (2) Immunogenic polypeptide: Human-derived AQP4 peptide segment p135 - 153 with the sequence LVTPPSVVGGLGVTMVHGN, and the polypeptide purity > 95%.

3. The method according to claim 1, wherein The step 2) of establishing a mouse model includes: (1) Weigh 1 mg of AQP4 peptide segment, dissolve it in 20 μl of DMSO, and then mix it with 980 μl of D-PBS buffer; (2) Weigh 3 mg of inactivated Mycobacterium tuberculosis and mix it with 1 ml of CFA; (3) Mix the above solutions in a 1:1 ratio, and repeatedly push and pull with a syringe on ice for 1 hour to prepare an oil-in-water emulsion; (4) Anesthetize the donor mice with isoflurane and subcutaneously inject the emulsion at 4 points on the back of the mice, 50 μl per point, 200 μl per mouse; (5) Eleven days after modeling, sacrifice the donor mice for the next experiment.

4. The method according to claim 1, wherein The step 3) of isolating spleen and lymph node cells from mice includes: (1) Decapitate the donor mice and soak them in 75% ethanol; (2) In a laminar flow hood, remove the spleen and lymph nodes (cervical, brachial plexus, axillary, and inguinal) of the mice and place them in EP tubes containing RPMI 1640 medium on ice respectively; (3) Place a 70 μm sieve in a 60 mm petri dish, add 5 ml of RPMI 1640 medium, gently grind the lymph nodes with the inner core of a 5 ml syringe, continuously suck the medium to rinse the sieve, and transfer the lymph node cell suspension to a 15 ml centrifuge tube; (4) Place a 70 μm sieve in a 60 mm petri dish, add 5 ml of mouse lymphocyte separation solution, gently grind the spleen with the inner core of a 5 ml syringe, continuously suck the separation solution to rinse the sieve, transfer the spleen cell suspension to a 15 ml centrifuge tube, and cover it with 1 ml of RPMI1640 medium to keep the liquid interface distinct; (5) Centrifuge the lymph node cell suspension at 1000 rpm at room temperature for 5 minutes; (6) Set the parameters 3, 2, and centrifuge the spleen cell suspension at 800 g at room temperature for 20 minutes; (7) Aspirate the lymphocyte layer, add 5 ml of RPMI 1640 medium to resuspend, and centrifuge at 1000 rpm at room temperature for 5 minutes; (8) Discard the supernatant, resuspend with an appropriate amount of flow cytometry buffer and count.

5. The method according to claim 1, wherein The step 4) of polarizing and identifying Tfh cells in mouse lymph node cells by flow cytometry includes: (1) Resuspend in 5 ml of lymphocyte culture medium and transfer to a 60 mm culture dish. Add mouse IL-6 (final concentration 20 ng / ml), IL-21 (final concentration 20 ng / ml), anti-IFN-γ monoclonal antibody (final concentration 10 μg / ml), anti-IL-4 monoclonal antibody (final concentration 10 μg / ml), and anti-TGF-β monoclonal antibody (final concentration 20 μg / ml); (2) Incubate in a 37°C, 5% CO2 cell culture incubator for 3 days; (3) After 3 days, take a small amount of cell suspension into a 5 ml flow tube, add 1 ml of flow buffer, and centrifuge at 1000 rpm at room temperature for 5 minutes; (4) Discard the supernatant, resuspend in 100 μl of flow buffer, add 0.5 μl of Zombie Aqua dead / live cell dye, mix well, and incubate at 4°C in the dark for 30 minutes; (5) Add 1 ml of flow buffer, centrifuge at 1000 rpm at room temperature for 5 minutes; (6) Discard the supernatant, resuspend in 100 μl of flow buffer, add 3 μl each of anti-mouse CD3-FITC, CD4-BV421, CD45-APC-Cy7, CD11b-APC, CXCR5-PE-Cy7, ICOS-PE, and PD1-BV605, mix well, and incubate at 4°C in the dark for 30 minutes; (7) Add 1 ml of flow buffer, centrifuge at 1000 rpm at room temperature for 5 minutes; (8) Discard the supernatant, resuspend in 250 μl of flow buffer and load onto the flow cytometer.

6. The method according to claim 1, wherein The sorting and flow cytometry identification of mouse total B cells in step (5) include: (1) Aspirate approximately 1x10 8 spleen cells into a 5 ml flow tube and resuspend with 1 ml of flow buffer; (2) Add 50 μl of rat serum; (3) Add 50 μl of Cocktail, mix, and incubate at room temperature for 5 minutes; (4) Vortex RapidSpheres for 30 seconds, add 50 μL of RapidSpheres, mix, and incubate at room temperature for 2.5 minutes; (5) Add flow buffer to 2.5 ml, pipette up and down several times to mix, place the flow tube in the magnetic pole, and incubate at room temperature for 2.5 minutes; (6) Pick up the magnetic pole, invert the magnet and the flow tube, and pour the enriched cell suspension into a new flow tube; (7) Take a small amount of cell suspension, add 5 μl of anti-mouse CD19-PE, mix well, and incubate at 4°C in the dark for 30 minutes; (8) Add 1 ml of flow buffer, centrifuge at 1000 rpm at room temperature for 5 minutes; (9) Discard the supernatant, resuspend in 250 μl of flow buffer and load onto the flow cytometer.

7. The method according to claim 1, characterized in that (7) The adoptive transfer of Tfh cells and total B cells in step (6) includes: (1) Aspirate approximately 2×10 7 Tfh cells or total B cells into an EP tube, and add flow cytometry buffer to make a suspension of 0.2 ml; (2) Aspirate the above cell suspension with a 1 ml insulin syringe and inject it into the recipient mice via the tail vein at a dose of 0.2 ml / animal. The control group is injected with 0.2 ml of flow buffer via the tail vein per animal; (3) Inject PTX into the recipient mice via the intraperitoneal route at a dose of 200 ng / animal / time on days 0 and 2; The Tfh cell group is the experimental group, the B cell group is the isotype control, and the control group is the negative control.

8. The method according to claim 1, wherein (7) The phenotypic assessment in step (7) includes: (1) Behavioral assessment: From the 5th day after adoptive transfer, the behavior of the mice was evaluated daily. The modified Kono's 5-point scoring criterion was used: 0 points: no disease; 0.5 points: partial weakness of the tail or slightly abnormal gait; 1.0 points: complete weakness of the tail or both partial weakness of the tail and slightly abnormal gait; 1.5 points: complete weakness of the tail and slightly weak hind limbs; 2.0 points: complete weakness of the tail and moderately weak hind limbs (scoring criterion: when climbing over the bars of the cage, the hind limbs frequently drop down); 2.5 points: need to drag the hind limbs to move; 3.0 points: complete paralysis of the hind limbs; 3.5 points: complete paralysis of the hind limbs and slightly weak forelimbs; 4.0 points: complete paralysis of all four limbs, only the head can move; 4.5 points: on the verge of death; 5.0 points: death; (2) Body weight and change value: From the 5th day after adoptive transfer, the mice were weighed daily, and the change value compared to the initial body weight on the 0th day was calculated.

9. The method according to claim 1, characterized in that, (2) The tissue sample preparation described above includes: (1) At the peak of disease onset, mice were intraperitoneally injected with 0.2 ml of 4% chloral hydrate. (2) After anesthetizing the mice, they were laid flat on their backs and fixed on a mouse board, and the abdomen was disinfected with 75% ethanol. (3) The scissors were used to cut open the bilateral chest walls along the lower edge of the costal arch, and the bilateral chest walls were turned upwards to expose the heart. The pericardium was removed with forceps, and an appropriate amount of D-PBS buffer was dropped to keep it moist. (4) A butterfly needle was inserted into the apex of the left heart, and 20 ml of cold D-PBS buffer was perfused. At the same time, the right auricle was cut open to drain the blood, and the color of the liver was observed to gradually turn white. (5) 20 ml of cold 4% paraformaldehyde was perfused. After the mice stopped twitching completely and all the tissues and organs of the whole body became hard. (6) Decapitation was performed, and the brain, lumbar enlargement of the spinal cord, and the left optic nerve were quickly removed. The above tissues were placed in 4% paraformaldehyde at 4°C and fixed overnight. (7) After washing with water, the above tissues were placed in a 15 ml centrifuge tube. (8) Dehydration: 70% alcohol for 1 hour → 85% alcohol for 1 hour → 90% alcohol for 1 hour → 95% alcohol for 0.5 hour → absolute ethanol (I) for 20 minutes → absolute ethanol (II) for 20 minutes; (9) Clearing: absolute ethanol + xylene (1:1) for 10 minutes → xylene (I) for 10 minutes → xylene (II) for 7 minutes; (10) Impregnation with paraffin and embedding: paraffin (I) (60°C) for 1 hour → paraffin (II) (60°C) for 1 hour → paraffin (III) (60°C) for 1 hour; (11) Sectioning: According to the atlas, sections of the striatum layer of the brain, the third ventricle / hippocampus layer of the brain, the transverse section of the lumbar enlargement of the spinal cord, and the longitudinal section of the optic nerve were cut out in sequence. (12) Mounting: The sections were put into a warm water bath. Under the action of surface tension, the sections were naturally flattened. The adhesive-free glass slides were tilted and inserted into the water surface to pick up the sections, so that the sections adhered to the glass slides. After being placed in an oven at 60°C for 2 hours, they were reserved for use.

10. The method according to claim 1, wherein (9) Histological HE staining described above includes: (1) Take out the sections, dewax to water: xylene (I) for 5 minutes → xylene (II) for 5 minutes → absolute ethanol (I) for 5 minutes → absolute ethanol (II) for 5 minutes → 95% ethanol for 5 minutes → 85% ethanol for 5 minutes → 75% ethanol for 5 minutes → 50% ethanol for 5 minutes → pure water for 5 minutes; (2) Stain with hematoxylin staining solution at room temperature for 5 minutes, and rinse with tap water for 10 minutes; (3) Differentiate with differentiating solution for 30 seconds, and rinse with tap water for 10 minutes; (4) Stain with eosin staining solution at room temperature for 2 minutes, and rinse with tap water for 10 minutes; (5) Dehydration and clearing: 95% ethanol (I) for 2 - 3 seconds → 95% ethanol (II) for 2 - 3 seconds → absolute ethanol for 5 minutes → absolute ethanol + xylene (1:1) for 5 minutes → xylene (I) for 2 minutes → xylene (II) for 2 minutes; (6) Mount the slide with neutral balsam, observe under the microscope and take pictures.

11. The method according to claim 1, characterized in that, (10) Histological LFB myelin staining includes: (1) Take out the section, dewax to water: xylene (I) for 5 minutes → xylene (II) for 5 minutes → absolute ethanol (I) for 5 minutes → absolute ethanol (II) for 5 minutes → 95% ethanol for 5 minutes → 85% ethanol for 5 minutes → 75% ethanol for 5 minutes → 50% ethanol for 5 minutes → pure water for 5 minutes; (2) Incubate with Luxol fast blue staining solution overnight at room temperature; (3) Wash off the excess staining solution with 95% ethanol, and rinse with tap water for 10 minutes; (4) Differentiate with Luxol differentiating solution for 15 seconds; (5) Differentiate with 70% ethanol for 30 seconds, rinse with tap water for 10 minutes, and observe under the microscope until the outlines of gray matter and white matter are distinct; (6) Counterstain with cresyl violet staining solution at room temperature for 30 seconds, and rinse with tap water for 10 minutes; (7) Dehydration and clearing: 95% ethanol (I) for 2 - 3 seconds → 95% ethanol (II) for 2 - 3 seconds → absolute ethanol for 5 minutes → absolute ethanol + xylene (1:1) for 5 minutes → xylene (I) for 2 minutes → xylene (II) for 2 minutes; (8) Mount the slide with neutral balsam, observe under the microscope and take pictures.

12. The method according to claim 1, wherein (11) Immunohistochemistry includes: (1) Take out the section, dewax to water: xylene (I) for 5 minutes → xylene (II) for 5 minutes → absolute ethanol (I) for 5 minutes → absolute ethanol (II) for 5 minutes → 95% ethanol for 5 minutes → 85% ethanol for 5 minutes → 75% ethanol for 5 minutes → 50% ethanol for 5 minutes → pure water for 5 minutes: (2) Boil the sodium citrate / EDTA antigen retrieval solution in a microwave oven → put the section in → high fire for 5 minutes, cool for 10 minutes, repeat 2 - 3 times, and cool naturally; (3) Add 3% hydrogen peroxide solution and incubate the section at room temperature for 10 minutes: (4) Wash with D-PBS buffer 3 times, 5 minutes each time: (5) Add 10% BSA and block at room temperature in a humidified box for 1 hour: (6) Discard the blocking solution, draw a hydrophobic circle 2 - 3 mm away from the sample edge and dry for 1 minute: (7) Dilute the primary antibody at 1:1000 and add it to the section, add 50 μl of antibody to each tissue, and incubate overnight at 4°C in a humidified box: (8) Wash with D-PBS buffer 3 times, 5 minutes each time: (9) Dilute the secondary antibody at 1:1000 and add it to the section, add 50 μl of antibody to each tissue, and incubate at room temperature for 30 minutes in a humidified box: (10) Wash with D-PBS buffer 3 times, 5 minutes each time: (11) Prepare the AB mixture, add 50 μl to each tissue, and incubate at room temperature for 3 hours in a humidified box: (12) Wash with D-PBS buffer 3 times, 5 minutes each time: (13) Prepare DAB staining solution, add 50 μl to each tissue, control the color development under the microscope, and rinse with tap water for 10 minutes: (14) Stain with hematoxylin staining solution at room temperature for 1 - 2 minutes, and rinse with tap water for 10 minutes: (15) Dehydration and clearing: 95% ethanol (I) for 2 - 3 seconds → 95% ethanol (II) for 2 - 3 seconds → absolute ethanol for 5 minutes → absolute ethanol + xylene (1:1) for 5 minutes → xylene (I) for 2 minutes → xylene (II) for 2 minutes. (16) Mount the slide with neutral balsam, observe under the microscope and take pictures.

13. The method according to claim 1, characterized in that, Use of the established adoptive transfer model of neuromyelitis optica spectrum disorder mice in the preparation of tools for screening therapeutic targets for neuromyelitis optica spectrum disorder.

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