A method for establishing a mouse model of Sjögren's syndrome

Abstract

The present invention relates to a method for establishing a mouse model of Sjögren's syndrome. The mouse model is established by injecting an emulsion prepared by mixing thymus protein with Titermax adjuvant into the sole of a mouse, and then performing water intake detection, saliva volume detection, tear volume detection, corneal fluorescein staining detection, histopathological detection and serum immunological detection on the mouse model, thereby screening and obtaining a qualified mouse model. This method only requires one immunization to complete the construction of the model, with little secondary damage, a short experimental cycle and a high model success rate. At the same time, the model is comprehensively and reliably verified and evaluated for relevant characteristic changes based on both in vivo and in vitro levels, providing a new tool and platform for the disease research of Sjögren's syndrome.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for establishing a Sjögren's syndrome mouse model. Background Art

[0002] Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease that mainly invades the exocrine glands (mainly salivary glands and lacrimal glands). The lymphocytic infiltration of the glands and the resulting autoimmune epithelial inflammation damage the secretions of the salivary glands and lacrimal glands. Its main clinical manifestations are dry mouth and dry eyes. In addition, 30%-40% of patients have systemic manifestations, involving the structure and function of organs such as the kidneys, lungs, joints and skin, as well as the nervous system. In addition, a variety of autoantibodies such as anti-Ro / SSA, anti-La / SSB, and anti-α-fodrin (fodrin) antibodies are present in the serum of patients and are significantly associated with the development of the disease. pSS mainly affects middle-aged women, but can also be observed in children, men and the elderly. The prevalence of the disease varies greatly, ranging from 0.01% to more than 3% of the total population, and the incidence ratio of women to men is 9:1, and the risk of female patients increases after menopause. In addition, primary Sjögren's syndrome occurs in the absence of other autoimmune diseases, while secondary Sjögren's syndrome (sSS) occurs on the basis of other potential autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and systemic sclerosis (SSc), combined with Sjögren's syndrome. The diagnosis of pSS is based on the comprehensive judgment of oral and ocular dryness symptoms and their evaluation (such as Schirmer test), focal lymphocytic infiltration in lip biopsy, and detection of autoantibodies in serum (such as anti-Ro / SSA).

[0003] The etiology of Sjögren's syndrome is multifactorial, with complex interactions between genetic, hormonal, environmental and immune risk factors. Although there has been some progress in the understanding of the pathogenesis of pSS, including the understanding of disease heterogeneity, the study of genes related to the disease, and the exploration of disease-related cellular pathways, the specific mechanism of Sjögren's syndrome is still unclear. Animal models are an important tool to help study human diseases and can help study the etiology and pathogenesis of diseases. To date, researchers have established multiple animal models of Sjögren's syndrome, including spontaneous models and immune-induced models. Spontaneous animal models have a great degree of similarity with the clinical manifestations of human Sjögren's syndrome, which can explain the effects of genetic factors and some environmental factors on the disease. However, spontaneous Sjögren's syndrome in mice usually occurs in specific mouse strains, such as transgenic or gene knockout mice, so the application of spontaneous models is severely limited. There are many autoantibodies in patients with Sjögren's syndrome, and some autoantibodies have certain specificity in Sjögren's syndrome and are closely related to the pathogenesis of the disease. The immune-induced model is to induce mice to produce characteristic antibodies of Sjögren's syndrome by combining autoantigens with immune adjuvants to enhance local immune responses, thereby establishing this research model. This model can make up for the shortcomings of spontaneous model research, but its disadvantages are that it requires many immunization times and a long modeling time, and the symptoms of the model mice are not obvious or are only confined to the salivary glands. At the same time, due to the long cycle, as the model mice age, various aspects of the body function decline, and the accuracy and stability of the experiment will also be affected. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for establishing a Sjögren's syndrome mouse model.

[0005] The technical scheme adopted by the present invention is: a method for establishing a Sjögren's syndrome mouse model, taking the thymus of a normal mouse, extracting thymic protein and then mixing it with a Titermax adjuvant to obtain a thymic protein-Titermax emulsion, and injecting the thymic protein-Titermax emulsion into a mouse to construct a Sjögren's syndrome mouse model.

[0006] Preferably, the specific steps are as follows:

[0007] Step 1: Take the thymus of a normal mouse, cut it into pieces and homogenize it thoroughly, take the supernatant after centrifugation, and adjust the thymus protein concentration to 2 mg / ml with PBS;

[0008] Step 2: 2 mg / ml thymus protein was mixed with an equal amount of Titermax adjuvant to prepare an emulsion to obtain 1 mg / ml thymus protein-Titermax emulsion;

[0009] Step 3: On day 0, the prepared 1mg / ml thymus protein-Titermax emulsion was injected into the midline of the sole of the mouse, slightly above the ankle. The injection volume was 0.1ml / mouse. After 12 weeks of modeling, the mice with successful modeling were screened by detecting indicators.

[0010] Preferably, in step 1, a normal C57BL / 6 mouse is killed by cervical dislocation, the mouse thymus is taken and placed in a pre-cooled PBS buffer, the capsule and connective tissue are stripped off, the thymus is cut into pieces, and fully homogenized in an ice bath, centrifuged at 12000g / 4°C for 30s, and then centrifuged at 3000g / 4°C for 15min, the supernatant is taken, the thymus protein concentration is measured by the BCA protein quantification method, and the thymus protein concentration is adjusted to 2 mg / ml with PBS.

[0011] Preferably, the detection index in step three includes one or more combinations of water intake detection, saliva volume detection, tear volume detection, corneal fluorescein staining detection, histopathological detection and serum immunological detection.

[0012] Preferably, the detection steps are as follows:

[0013] The drinking water volume detection method is to first weigh the drinking water bottle filled with water, and then weigh the weight of the remaining water in the drinking water bottle again after one week. The difference between the two is the drinking water volume of the mice in one week;

[0014] Saliva volume detection: The method is to fast the mice for 16 hours before the measurement, and use an animal anesthesia machine for deep anesthesia, with stable breathing, disappearance of corneal reflex, and relaxation of limb muscles as the anesthesia standard; after complete anesthesia, the mice are intraperitoneally injected with 0.5mg / kg body weight pilocarpine solution and a pre-weighed cotton ball is stuffed into the mouse's mouth, which is taken out after 20 minutes, and the mouse's saliva volume is calculated using the wet weight method;

[0015] Tear volume detection: The method is to fast the mice for 16 hours before measurement, use an animal anesthesia machine for deep anesthesia, and use steady breathing, disappearance of corneal reflex, and relaxation of limb muscles as the anesthesia standard; after complete anesthesia, the mice are intraperitoneally injected with 0.5mg / kg body weight pilocarpine solution, wait for 10 minutes, put the Schirmer test paper on the outer canthus of the mouse, and take it out after 10 minutes. The length of the discolored part of the test paper is the tear volume;

[0016] Corneal fluorescein staining: performed on day 0 and week 12, using a micropipette to drip physiological saline into the conjunctival sac below the C57BL / 6 mice without surface anesthesia for flushing, and then using a micropipette to drip 1 μL of 1% sodium fluorescein solution, and then examined under a slit lamp microscope with cobalt blue light and photographed and recorded 3 minutes later. Dot-like or flake-like staining appeared on the surface of the mouse cornea after week 12 of modeling;

[0017] Histopathological examination was conducted at the 13th week. The method was to take out 2 to 3 mice from the experimental group and the control group, take blood from the eyeballs and then kill them by dislocating the neck. The lacrimal glands and salivary glands were taken for pathological examination. The lacrimal glands and salivary glands should be fixed in 4% paraformaldehyde solution for 24 hours, and then washed, dehydrated, embedded, sliced ​​and fished for H&E staining. There was obvious lymphocyte infiltration in the lacrimal glands and salivary glands of the experimental group, but no obvious infiltration in the control group.

[0018] Serum immunological test was conducted at week 13. The method was to collect serum from mice in the experimental group and the control group, respectively, and use enzyme-linked immunosorbent assay (ELISA) to detect autoantibodies. The serum was prepared by removing the eyeballs of mice and then placing the collected blood at 4° overnight. The collected blood was centrifuged at 500g for 20 minutes to separate the serum. The upper layer of serum was transferred to a new centrifuge tube and placed at -20°C for use. A large amount of autoantibodies appeared in the serum of the experimental group, while no autoantibodies appeared in the control group.

[0019] Preferably, the mouse is a C57BL / 6 mouse, preferably a female C57BL / 6 mouse of 3-4 weeks old.

[0020] The invention relates to a method for establishing a mouse model of Sjögren's syndrome and application of the mouse model constructed in screening drugs for treating and / or preventing Sjögren's syndrome.

[0021] Preferably, the drug to be tested is administered to the mouse model constructed by the construction method described in any one of claims 1 to 6; the therapeutic effect of the drug to be tested is analyzed and evaluated, and the test drug that can significantly improve the symptoms of Sjögren's syndrome in the mouse model is selected.

[0022] A method for evaluating a mouse model of Sjögren's syndrome, wherein after establishing the mouse model, the mouse model is subjected to water intake detection, saliva volume detection, tear volume detection, corneal fluorescein staining detection, histopathological detection and serum immunological detection;

[0023] Among them, histopathological examination was performed 10-13 weeks after modeling, and if obvious lymphocyte infiltration was detected, the prepared mouse model was judged to be a qualified mouse model and could be used for subsequent experiments.

[0024] Preferably, the mouse model testing criteria also include:

[0025] Water intake test: Start testing on day 0 and test once a week; water intake begins to increase after 5-6 weeks of modeling;

[0026] Saliva volume test: Start testing on day 0 and test every 1-3 weeks; the saliva volume gradually decreases after the 8th to 9th week of modeling, and is lower than normal after the 11th to 12th week;

[0027] Tear volume detection: Start detection on day 0 and detect once every 1-3 weeks; Tear volume gradually decreases after 8-9 weeks of modeling, and is lower than normal after 11-12 weeks;

[0028] Corneal fluorescein staining test: performed on day 0 and week 12; after week 12 of modeling, dot-like or flake-like staining appeared on the surface of the mouse cornea;

[0029] Serum immunological test: performed from 10th to 13th week, a large amount of autoantibodies appear in the serum.

[0030] The advantages and positive effects of the present invention are: a single injection of an emulsion prepared by mixing Titermax adjuvant and thymus protein can complete the construction of a Sjögren's syndrome model, the experimental period is short, the modeling rate is high, and it can be carried out on mice that have not been genetically modified. Compared with previous models, the efficiency of modeling is greatly improved; the mouse model only needs one immunization to avoid damage to the mouse's mental and functional state caused by multiple injections, thereby improving the stability of the modeling;

[0031] Based on the symptoms, signs, immune and pathological manifestations of Sjögren's syndrome, a standardized evaluation system suitable for Sjögren's syndrome animal model has been established. Standard and comprehensive testing methods will make the experimental results have a certain degree of accuracy, comparability and repeatability, which will be conducive to the research and exploration of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The workflow of the present invention is:

[0033] Figure 2 The changes in weekly water intake of mice in the experimental and control groups (* represents p<0.05, ** represents p<0.01);

[0034] Figure 3 The changes in saliva volume of mice in the experimental group and the control group on day 0, week 6, week 9, and week 12 (* represents p < 0.05);

[0035] Figure 4 The changes in tear volume of mice in the experimental group and the control group at day 0, week 6, week 9, and week 12 (* represents p < 0.05);

[0036] Figure 5 The corneal fluorescein staining of mice in the experimental group and the control group on day 0 and week 12 (taken from the visual field under a slit lamp 400x light microscope);

[0037] Figure 6 The histopathological examination (HE staining) of mice in the experimental group and the control group (all taken from the field of view under a 200x light microscope);

[0038] Figure 7The results of serum immunological test (ELISA) of mice in the experimental group and the control group (* represents p<0.05, ** represents p<0.01, *** represents p<0.001, **** represents p<0.0001). DETAILED DESCRIPTION

[0039] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0040] The present invention relates to a method for establishing and evaluating a mouse model of Sjögren's syndrome, which can quickly establish a mouse model. The thymus of a normal mouse is taken, and an emulsion prepared by mixing thymus protein of a C57BL / 6 mouse with a Titermax adjuvant is injected into the midline of the sole of the foot of the experimental group of mice at the ankle, with the needle inserted about 3 mm, 50ul for each sole, and 100ul for the two hind soles. The specific construction method and process are as follows: Figure 1 As shown. Through the antigen-antibody immune reaction, the exocrine glands are induced to produce a large number of lymphocyte infiltrations and dry symptoms and signs in mice. Thus, a mouse model of Sjögren's syndrome is constructed. This establishment method is based on the thymus protein of normal C57BL / 6 mice. The Titermax adjuvant is used to assist the protein antigen to immunize mice. Through the interaction between antigen and antibody, the body's inflammatory response and corresponding immune response are induced, and then the Sjögren's syndrome animal model is induced. The construction of the Sjögren's syndrome model can be completed by a single injection of an emulsion prepared by mixing Titermax adjuvant and thymus protein, which greatly improves the shortcomings of the previous model with a long modeling cycle and a large number of immunizations, and reduces the secondary damage to the model animals.

[0041] In addition, an accurate and reliable model is the basic premise for continuing the next step of research. Based on the symptoms, signs, immune and pathological manifestations of Sjögren's syndrome, the present invention systematically evaluates the animal model in vivo and in vitro. The manifestation of Sjögren's syndrome symptoms and signs is tested by water intake, saliva and tear secretion, and corneal staining, and the model is qualitatively tested based on serum-specific antibodies and pathological staining tests. A comprehensive and effective evaluation method is provided. The constructed mice can provide a good tool and platform for studying the pathogenesis of the disease and evaluating treatment options.

[0042] The scheme of the present invention is described below in conjunction with the accompanying drawings, wherein the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.

[0043] Among them, thymic proteins were derived from fresh thymus tissue of normal C57BL / 6 mice obtained under sterile conditions;

[0044] Titermax adjuvant is a commercial product purchased from Sigma-Aldrich, USA, with the product number Cat# H4397;

[0045] C57BL / 6 mice were purchased from Nanjing Laboratory Animal Research Institute.

[0046] Example 1: Construction of mouse model

[0047] 1.1 Preparation of thymus protein-Titermax emulsion:

[0048] A normal C57BL / 6 mouse was killed by dislocating the neck, disinfected with 75% alcohol, and the mouse thymus was removed under aseptic conditions in a clean bench. It was then immediately placed in a culture dish with pre-cooled PBS buffer, the capsule and connective tissue were peeled off on ice, washed with PBS and placed in a sterile enzyme-free centrifuge tube, the thymus in the tube was cut into pieces using tissue scissors, and fully homogenized using a homogenizer in an ice bath, centrifuged at 12000g / 4℃ for 30s, and then at 3000g / 4℃ for 15min, the supernatant was taken, and BCA protein was used for determination. Measure the thymic protein concentration by the titration method. Adjust the thymic protein concentration to 2 mg / ml with PBS. Suck 2 mg / ml thymic protein and an equal amount of Titermax adjuvant into two syringes respectively. Connect the syringe with adjuvant to a connector. Push the syringe slowly to exhaust the air in the connector. Then connect the connector to another syringe with thymic protein. Make sure to exhaust all the air. Then push the syringes alternately until a viscous emulsion is formed. This is to prepare a 1 mg / ml thymic protein-Titermax emulsion.

[0049] 1.2 Preparation of PBS-Titermax emulsion:

[0050] Aspirate the sterile PBS solution and an equal amount of Titermax adjuvant into two syringes respectively, connect the syringe with the adjuvant to a connector, slowly push the syringe to exhaust the air in the connector, and then connect the connector to another syringe with PBS, making sure to exhaust all the air, and then push the syringes alternately until a viscous emulsion is formed. This is how the PBS-Titermax emulsion is prepared.

[0051] 1.3 Random grouping of C57BL / 6 mice:

[0052] Thirty C57BL / 6 mice were weighed and then randomly divided into an experimental group and a control group, with 15 mice in each group. The experimental group was numbered with ear tags, and the control group was not treated to ensure that the number, size, weight, and condition of mice in each group were as similar as possible.

[0053] 1.4 Immunization of mice:

[0054] On day 0, mice were deeply anesthetized using an animal anesthesia machine, with steady breathing, disappearance of corneal reflex, and relaxation of limb muscles as the anesthesia standard; after complete anesthesia, the soles of the mice were disinfected with 75% alcohol, and the needle was inserted into the sole with the needle tip of the syringe downward at the ankle on the midline of the sole, about 3 mm deep, and 50ul of thymus protein-Titermax emulsion was subcutaneously immunized in each sole, with a total of 100ul for the immunization of both hind soles; the mice in the control group were injected with an equal amount of PBS-Titermax emulsion using the same method.

[0055] Example 2: Detection indicators

[0056] The mouse model prepared in Example 1 was used to perform water intake detection, saliva volume detection, tear volume detection, corneal fluorescein staining detection, histopathological detection and serum immunological detection on the experimental group and the control group, respectively. The detection methods and standards are as follows.

[0057] (1) Water intake test: Start the test on day 0 and measure once a week. The method is to weigh the water bottle filled with water first, and then weigh the weight of the remaining water in the water bottle again after one week. The difference between the two is the water intake of the mouse in one week. Qualified mouse models have a trend of gradually increasing water intake after the 5th to 6th week of modeling;

[0058] The experimental group and the control group mice were tested respectively. Figure 2 As shown, the experimental and control group mice showed differences in water drinking starting from the 6th week, and the experimental group mice drank significantly more water than the control group mice.

[0059] (2) Saliva volume detection: The test was conducted on day 0, week 6, week 9, and week 12. The method was to fast the mice for 16 hours before the test, and then use an animal anesthesia machine for deep anesthesia, with steady breathing, disappearance of corneal reflex, and relaxation of limb muscles as the anesthesia standard; after complete anesthesia, the mice were weighed using an electronic balance, 0.5 mg / kg body weight of pilocarpine solution was injected into the mouse's peritoneum, and a pre-weighed cotton ball was stuffed into the mouse's mouth and removed after 20 minutes. The wet weight method was used to calculate the weight of the mouse's saliva; the qualified mouse model had a tendency to gradually decrease in saliva volume after the 8th to 9th week of modeling, and was lower than normal after the 11th to 12th week;

[0060] The experimental group and the control group mice were tested respectively. Figure 3 As shown, it can be seen that the saliva secretion of mice in the experimental group is lower than that of mice in the control group, and the difference gradually increases with time.

[0061] (3) Tear volume detection: The test was conducted on day 0, week 6, week 9, and week 12. The method was to fast the mice for 16 hours before the test, and then use an animal anesthesia machine for deep anesthesia, with steady breathing, disappearance of corneal reflex, and relaxation of limb muscles as the anesthesia standard; after complete anesthesia, the mice were weighed using an electronic balance, and 0.5 mg / kg body weight of pilocarpine solution was injected intraperitoneally into the mice. After waiting for 10 minutes, the Schirmer test paper was placed on the outer canthus of the mice and removed after 10 minutes. The length of the discolored part of the test paper was the tear volume; the qualified mouse model showed a gradual decrease in tears from the 8th to 9th week of modeling, and was lower than normal after the 11th to 12th week;

[0062] The experimental group and the control group mice were tested respectively. Figure 4 As shown, the tear secretion of mice in the experimental group was lower than that of mice in the control group, and the difference gradually increased with time.

[0063] (4) Corneal fluorescein staining test: It was performed on day 0 and week 12 by using a micropipette to drip normal saline into the conjunctival sac below the C57BL / 6 mouse without surface anesthesia for flushing, and then using a micropipette to drip 1 μL of 1% sodium fluorescein solution. After 3 minutes, the cornea was examined under a slit lamp microscope using cobalt blue light and photographed. Qualified mouse models showed punctate or flake staining on the surface of the mouse cornea 11-12 weeks after modeling.

[0064] After 12 weeks of modeling, the mice in the experimental group and the control group were tested respectively. Figure 5 As shown, there was no change on the corneal surface of the mice in the control group, while there was flake staining on the corneal surface of the mice in the experimental group.

[0065] (5) Histopathological examination: It was conducted at the 13th week. Two to three mice were taken from the experimental group and the control group respectively. After blood was collected from the eyeballs, the mice were killed by dislocating the neck. The lacrimal glands and salivary glands were taken for pathological examination. The lacrimal glands and salivary glands were fixed in 4% paraformaldehyde solution for 24 hours, and then washed, dehydrated, embedded, sliced, and fished for H&E staining. The lacrimal glands and salivary glands of qualified mouse models should have obvious lymphocyte infiltration, while the control group had no obvious infiltration.

[0066] After 13 weeks of modeling, the mice in the experimental group and the control group were tested respectively, and the thymus, spleen, lacrimal gland and parotid gland tissue sections of the mice were stained and tested. Figure 6 As shown, lymphocyte infiltration can be seen in the lacrimal gland and parotid gland tissue sections of the experimental group mice.

[0067] (6) Serum immunological test: It was conducted at the 13th week. The method was to collect serum from the experimental group and the control group mice respectively, and use enzyme-linked immunosorbent assay (ELISA) to detect autoantibodies. The serum was prepared by removing the eyeballs of the mice with ophthalmic forceps to collect blood. The collected blood was placed at 4°C overnight, and then centrifuged at 500g for 20 minutes to separate the serum. The upper layer of serum was transferred to a new centrifuge tube and placed at -20°C for use. Qualified mouse model serum should show a large amount of autoantibodies, while the control group should not show autoantibodies.

[0068] After 13 weeks of modeling, the mice in the experimental group and the control group were tested respectively. Figure 7 As shown, there was a significant difference in serum antibodies between the experimental group mice and the control group mice, and the amount of antibodies in the serum of the experimental group mice was significantly higher than that of the conventional mice in the control group.

[0069] The above monitoring method and evaluation method evaluate the mouse model from the phenotypic, immune and pathological levels, respectively, so as to reduce the false positive rate of the model. It can be seen from the above indicators that the establishment method of the present invention can quickly construct a Sjögren's syndrome mouse model, and 11 of the 15 experimental group mice were finally measured to be modeled. The mouse modeling stability is high, and the mouse modeling success rate is higher than 70%, which can provide a tool for subsequent basic research and explore the possible mechanism of Sjögren's syndrome on the basis of this model.

[0070] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A method for establishing a Sjögren's syndrome mouse model, characterized in that: The mouse thymus protein was used as an antigen to immunize the mouse; the thymus of normal mice was taken, the thymus protein was extracted and mixed with Titermax adjuvant to obtain thymus protein-Titermax emulsion, and the thymus protein-Titermax emulsion was injected into the mouse to construct a Sjögren's syndrome mouse model; The specific steps are as follows: Step 1: Take normal C57BL / 6 mice and kill them by cervical dislocation. Take the mouse thymus and place it in pre-cooled PBS buffer. Remove the capsule and connective tissue, cut the thymus into pieces, fully homogenize it in an ice bath, take the supernatant after centrifugation, and adjust the thymus protein concentration to 2 mg / ml with PBS; Step 2: 2 mg / ml thymus protein was mixed with an equal amount of Titermax adjuvant to prepare an emulsion to obtain 1 mg / ml thymus protein-Titermax emulsion; Step 3: A single injection of the emulsion prepared by mixing Titermax adjuvant and thymus protein was given. On day 0, the prepared 1 mg / ml thymus protein-Titermax emulsion was injected into the midline of the sole of the mouse's foot, slightly above the ankle. The injection volume was 0.1 ml per mouse. After 12 weeks of modeling, the mice with successful modeling were screened by detecting indicators.

2. The method for establishing a mouse model of Sjögren's syndrome according to claim 1, characterized in that: In step 1, normal C57BL / 6 mice were killed by cervical dislocation, the mouse thymus was taken and placed in pre-cooled PBS buffer, the capsule and connective tissue were stripped off, the thymus was cut into pieces, and fully homogenized in an ice bath, centrifuged at 12000g / 4℃ for 30s, and then centrifuged at 3000g / 4℃ for 15min, the supernatant was taken, and the thymus protein concentration was measured by BCA protein quantification method, and the thymus protein concentration was adjusted to 2mg / ml with PBS.

3. The method for establishing a Sjögren's syndrome mouse model according to claim 1, characterized in that: The detection indicators in step three include one or more combinations of water intake detection, saliva volume detection, tear volume detection, corneal fluorescein staining detection, histopathological detection and serum immunological detection.

4. The method for establishing a mouse model of Sjögren's syndrome according to claim 3, characterized in that: The detection steps are as follows: Water intake test: First weigh the water bottle filled with water, and then weigh the water bottle and the remaining water again after one week. The difference between the two is the water intake of the mice in one week. Saliva volume detection: The mice were fasted for 16 hours before measurement and deeply anesthetized. The anesthesia standard was stable breathing, disappearance of corneal reflex, and relaxation of limb muscles. After complete anesthesia, 0.5 mg / kg body weight of pilocarpine solution was injected intraperitoneally into the mice, and a cotton ball weighed in advance was stuffed into the mouth of the mice. After 20 minutes, it was taken out and the saliva volume of the mice was calculated by wet weight method. Tear volume detection: The mice were fasted for 16 hours before measurement and deeply anesthetized, with stable breathing, disappearance of corneal reflex, and relaxation of limb muscles as the anesthesia standard; after complete anesthesia, the mice were intraperitoneally injected with 0.5 mg / kg body weight pilocarpine solution, waited for 10 minutes, and the Schirmer test paper was placed on the outer canthus of the mouse and taken out after 10 minutes. The length of the discolored part of the test paper represented the tear volume; Corneal fluorescein staining test: On day 0 and week 12, normal saline was dripped into the conjunctival sac of mice without surface anesthesia using a micropipette for flushing, and then 1 μL of 1% sodium fluorescein solution was dripped using a micropipette. After 3 minutes, the cornea was examined under a slit lamp microscope using cobalt blue light and photographed and recorded. The image results on day 0 and week 12 were compared. Histopathological examination: At the 13th week, 2-3 mice were taken from the experimental group and the control group, and blood was collected from the eyeballs and then the mice were killed by dislocating the neck. The lacrimal glands and salivary glands were taken for pathological examination. The lacrimal gland and salivary gland tissue sections were stained with H&E, and the image results of the experimental group and the control group were compared. Serum immunological test: At week 13, sera were collected from mice in the experimental and control groups, and autoantibodies were detected using enzyme-linked immunosorbent assay (ELISA).

5. The method for establishing a mouse model of Sjögren's syndrome according to claim 1, characterized in that: Mice were 3-4 week old female C57BL / 6 mice.

6. Use of the mouse model constructed by the method for establishing a Sjögren's syndrome mouse model according to any one of claims 1 to 5 in screening drugs for treating and / or preventing Sjögren's syndrome.

7. The use according to claim 6, characterized in that: The drug to be tested is administered to the mouse model constructed by the construction method according to any one of claims 1 to 5; the therapeutic effect of the drug to be tested is analyzed and evaluated, and the test drug that can significantly improve the symptoms of Sjögren's syndrome in the mouse model is selected.

8. A method for evaluating a mouse model of Sjögren's syndrome, characterized in that: A mouse model prepared by the method for establishing a mouse model of Sjögren's syndrome according to any one of claims 1 to 5; After the mouse model was established, the mouse model was tested for water intake, saliva volume, tear volume, corneal fluorescein staining, histopathology, and serum immunology; Among them, histopathological examination was performed 10-13 weeks after modeling, and if obvious lymphocyte infiltration was detected, the prepared mouse model was judged to be a qualified mouse model and could be used for subsequent experiments.

9. The method for evaluating a mouse model of Sjögren's syndrome according to claim 8, characterized in that: Mouse model testing criteria also include: Water intake test: Start testing on day 0 and test once a week; water intake begins to increase after 5-6 weeks of modeling; Saliva volume test: Start testing on day 0 and test every 1-3 weeks; the saliva volume gradually decreases after the 8th to 9th week of modeling, and is lower than normal after the 11th to 12th week; Tear volume detection: Start detection on day 0 and detect once every 1-3 weeks; Tear volume gradually decreases after 8-9 weeks of modeling, and is lower than normal after 11-12 weeks; Corneal fluorescein staining test: performed on day 0 and week 12; after week 12 of modeling, dot-like or flake-like staining appeared on the surface of the mouse cornea; Serum immunological test: performed from 10th to 13th week, a large amount of autoantibodies appear in the serum.

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