Hashimoto's thyroiditis model and construction method thereof, antigen

Abstract

The application discloses a Hashimoto's thyroiditis model, a construction method thereof and an antigen, and the construction method comprises the following steps: preparing a pig thyroid lyophilized powder; selecting 8-week-old EAT susceptible strain CBA / J female mice, and injecting phosphate buffered saline solutions containing the pig thyroid lyophilized powder at the 0th day, the 7th day and the 14th day after 8 weeks; injecting phosphate buffered saline solutions containing lipopolysaccharide at a predetermined time interval; when the EAT susceptible strain CBA / J female mice are 12 weeks old, the modeling effect is measured from thyroid pathological morphology and EAT scores, serum TgAb concentration, serum TPOAb concentration and the number of each T lymphocyte subpopulation in the spleen. After the mice are continuously injected with the phosphate buffered saline solutions containing the pig thyroid lyophilized powder and the lipopolysaccharide for three times through the tail vein, the mice produce autoantibodies, and obvious lymphatic infiltration appears in the thyroid tissue, which indicates that the pig thyroid lyophilized powder can induce the occurrence of Hashimoto's thyroiditis, and a new thought is provided for the pathogenesis of Hashimoto's thyroiditis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a Hashimoto's thyroiditis model, a construction method thereof and an antigen. BACKGROUND

[0002] Hashimoto's thyroiditis (HT) is also called chronic lymphocytic thyroiditis (CLT) or autoimmune thyroiditis (AIT), and is the autoimmune disease with the highest incidence at present, with a population incidence of 8 / 1000 to 46 / 1000. The main performance of HT patients is diffuse enlargement of the thyroid gland, a large number of lymphocyte infiltration in the thyroid gland, extensive destruction and atrophy of thyroid follicles, lymphoid germinal center fibrosis, and gradual conversion of the thyroid function of the patients to hypothyroidism or subclinical hypothyroidism, and significant increase of serum thyroid peroxidase antibody (TPOAb) and / or thyroglobulin antibody (TgAb) levels. HT is the most common cause of hypothyroidism in clinic, and can cause more than 90% of primary hypothyroidism. Meanwhile, the quality of life of HT patients is significantly lower than that of normal people, and the risk of suffering from cardiovascular and cerebrovascular diseases, metabolic syndrome, mental depression and thyroid cancer of the HT patients is also significantly increased.

[0003] At present, the pathogenesis of Hashimoto's thyroiditis is not completely clear, and therefore, it is necessary to construct a stable and efficient Hashimoto's thyroiditis model. SUMMARY

[0004] In order to solve the above problems existing in the prior art, the application provides a Hashimoto's thyroiditis model, a construction method thereof and an antigen. The technical problems to be solved by the application are solved by the following technical solutions.

[0005] The application provides a construction method of a Hashimoto's thyroiditis model, comprising the following steps:

[0006] S1: preparing a pig thyroid gland freeze-dried powder;

[0007] S2: selecting 8-week-old EAT susceptible strain CBA / J female mice, and injecting phosphate buffered saline solutions containing the pig thyroid gland freeze-dried powder at the 0th day, the 7th day and the 14th day after the 8th week, and injecting phosphate buffered saline solutions containing lipopolysaccharide at a predetermined time interval;

[0008] S3: At the age of 12 weeks, the EAT-susceptible strain CBA / J female mice are measured for the modeling effect from the thyroid pathological morphology and EAT score, serum TgAb concentration, serum TPOAb concentration and the number of each sub-group of spleen T lymphocytes.

[0009] In one embodiment of the present application, the S1 comprises:

[0010] S1.1: Separating thyroid tissue from the neck part of freshly slaughtered pigs;

[0011] S1.2: Removing connective tissue in the thyroid tissue in an ice bath, and grinding the thyroid tissue after removing the connective tissue by using a grinder to obtain a thyroid extract;

[0012] S1.3: Freezing the thyroid extract in a refrigerator overnight and placing it in liquid nitrogen for a predetermined time to pretreat the thyroid extract;

[0013] S1.4: Freeze-drying the pretreated thyroid extract to obtain a pig thyroid freeze-dried powder and freeze-preservation.

[0014] In one embodiment of the present application, the S1.1 comprises:

[0015] Separating thyroid tissue from the neck part of freshly slaughtered pigs, placing it in phosphate buffered saline at 4°C and transporting it to the laboratory, wherein the pH value of the phosphate buffered saline is 7.0, the concentration of phosphate in the phosphate buffered saline is 15 mMol / L, and the concentration of sodium chloride is 140 mMol / L.

[0016] In one embodiment of the present application, the S1.2 comprises:

[0017] Removing connective tissue in the thyroid tissue in an ice bath, and cutting the thyroid tissue after removing the connective tissue into thyroid tissue blocks of 0.1-0.2 g;

[0018] Removing connective tissue in the thyroid tissue in an ice bath, and cutting the thyroid tissue after removing the connective tissue into thyroid tissue blocks of 0.1-0.2 g;

[0019] Placing the thyroid tissue blocks in phosphate buffered saline and grinding them in a tissue grinder, the grinding temperature is 4°C, and then centrifuging at a speed of 3000 rpm / min for 15 min, wherein the volume ratio of the phosphate buffered saline to the thyroid tissue blocks is 10:1;

[0020] After the centrifugation is completed, the supernatant is recovered, which is the thyroid extract.

[0021] In one embodiment of the present application, after the S1, it further comprises:

[0022] In use, the porcine thyroid gland lyophilized powder is dissolved into a 10 ug / ul sterile porcine thyroid gland lyophilized powder solution using a phosphate buffered saline solution.

[0023] In an embodiment of the present application, the phosphate buffered saline solution containing porcine thyroid gland lyophilized powder is 100 μL of phosphate buffered saline solution containing 1000 μg of porcine thyroid gland lyophilized powder.

[0024] In an embodiment of the present application, the phosphate buffered saline solution containing lipopolysaccharide is 100 μL of phosphate buffered saline solution containing 100 μg of lipopolysaccharide.

[0025] Another aspect of the present application provides a Hashimoto's thyroiditis model constructed using the construction method of any one of the above embodiments.

[0026] Still another aspect of the present application provides an antigen for constructing a Hashimoto's thyroiditis model, the antigen being a 10 ug / ul sterile porcine thyroid gland lyophilized powder solution, and the active ingredients of the antigen being thyroid globulin and thyroid peroxidase.

[0027] In an embodiment of the present application, the antigen is an injection.

[0028] Compared with the prior art, the present application has the following beneficial effects:

[0029] 1. The present application provides a construction method of a Hashimoto's thyroiditis model. After a CBA / J female mouse of an EAT susceptible strain is injected with a phosphate buffered saline solution containing porcine thyroid gland lyophilized powder and a phosphate buffered saline solution containing lipopolysaccharide for three times in succession, the mouse produces autoantibodies, and obvious lymphatic infiltration appears in the thyroid tissue, indicating that the model construction is successful. The porcine thyroid gland lyophilized powder can induce the occurrence of Hashimoto's thyroiditis, and this modeling method provides a new train of thought for the pathogenesis of Hashimoto's thyroiditis.

[0030] 2. The present application confirms that Th17 (CD4+IL-17A+) and Treg (CD4+CD25+Foxp3+) cells in the mouse spleen play an important role in the pathogenesis of autoimmune thyroiditis in mice by detecting the cell proportion of Th17 (CD4+IL-17A+) and Treg (CD4+CD25+Foxp3+) in the mouse spleen. This finding provides a relatively solid theoretical basis and experimental data as support for future in-depth study of the immune tolerance mechanism of Hashimoto's thyroiditis.

[0031] The present application will be further described in detail below in combination with the drawings and embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is a flow chart of a construction method of a Hashimoto's thyroiditis model provided by an embodiment of the present application.

[0033] Figure 2 is a preparation flowchart of the pig thyroid lyophilized powder provided by the embodiment of the present application;

[0034] Figure 3 is an experimental flowchart of using the pig thyroid lyophilized powder to induce Hashimoto's thyroiditis in CBA / J female mice provided by the embodiment of the present application;

[0035] Figure 4 is a scatter plot of the thyroid HE staining of mice in each group and the thyroid inflammation pathological score of mice in each group;

[0036] Figure 5 is a column chart of the relative concentration of serum TgAb and a column chart of the relative concentration of TPOAb of mice in each group;

[0037] Figure 6 is a flow chart and a column chart of the proportion of Th17 and Treg cells in the spleen of mice in each group. DETAILED DESCRIPTION

[0038] In order to further illustrate the technical means and effects adopted by the present application to achieve the predetermined object, the following will be described in detail in combination with the drawings and specific embodiments, and the Hashimoto's thyroiditis model, the construction method thereof and the antigen according to the present application will be described in detail.

[0039] The foregoing and other technical contents, features and effects of the present application can be clearly presented in the following detailed description of specific embodiments in combination with the drawings. Through the description of the specific embodiments, the technical means and effects adopted by the present application to achieve the predetermined object can be understood more deeply and specifically. However, the attached drawings are provided for reference and illustration only, and are not used to limit the technical solutions of the present application.

[0040] It should be noted that, in this document, relational terms such as first and second, and the like, are used solely to distinguish one entity or action from another entity or action, without necessarily requiring or implying any such actual relationship or order between such entities or actions. Moreover, the terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element preceded by "comprises a" does not, without more constraints, foreclose the existence of additional identical elements in the process, method, article, or apparatus that comprises the recited element.

[0041] Embodiment one

[0042] Please refer to Figure 1 , Figure 1is a flow chart of a method for constructing a bridge-based Hashimoto's thyroiditis model provided by an embodiment of the present application. The method for constructing comprises the following steps:

[0043] S1: preparing a pig thyroid lyophilized powder.

[0044] As shown in Figure 2 , step S1 of the embodiment specifically comprises:

[0045] S1.1: separating thyroid tissue from the neck of a freshly slaughtered pig.

[0046] Specifically, the thyroid tissue is separated from the neck of a freshly slaughtered pig, placed in phosphate buffered saline (PBS) with a pH value of 7.0 and a phosphate concentration of 15 mMol / L and a sodium chloride (NaCl) concentration of 140 mMol / L at 4°C, and transported to the laboratory.

[0047] S1.2: removing connective tissue in the thyroid tissue in an ice bath, and grinding the thyroid tissue after the removal of the connective tissue using a grinder to obtain a thyroid extract.

[0048] Specifically, the connective tissue such as fat and envelope in the thyroid tissue is removed in an ice bath, and the thyroid tissue after the removal of the connective tissue is cut into thyroid tissue blocks of 0.1-0.2 g; the thyroid tissue blocks are placed in 10 volumes of phosphate buffered saline (i.e., the volume ratio of phosphate buffered saline to thyroid tissue blocks is 10:1) and ground in a low-temperature tissue grinder, the grinding temperature is 4°C, the grinding is performed twice, each for 60 seconds, the interval between the two grindings is 15 seconds, and the grinding power is 60 Hz. The low-temperature tissue grinder used in the embodiment has a model number of Jingxin JXFSTPRP-III. Subsequently, the thyroid extract is recovered after centrifugation at a speed of 3000 rpm / min for 15 min in a centrifuge.

[0049] S1.3: freezing the thyroid extract in a refrigerator overnight and placing it in liquid nitrogen for a predetermined time to pretreat the thyroid extract.

[0050] Specifically, the pretreatment process of the embodiment is that the obtained thyroid extract is placed in a refrigerator at-80°C overnight, and then the thyroid extract is placed in liquid nitrogen for at least 1 hour.

[0051] S1.4: freeze-drying the pretreated thyroid extract to obtain a pig thyroid lyophilized powder and freeze-preserve the powder.

[0052] Specifically, the pretreated thyroid extract is freeze-dried by a freeze dryer to obtain a freeze-dried pig thyroid powder. The freeze-dried pig thyroid powder is stored at -80°C for standby. The model of the freeze dryer used in this example is FDU-2110.

[0053] Further, in use, the freeze-dried pig thyroid powder is dissolved into a sterile freeze-dried pig thyroid powder solution of 10 ug / ul by using a phosphate buffered saline solution.

[0054] S2: Select 8-week-old EAT susceptible strain CBA / J female mice as a model group (pTLP group), and inject the EAT susceptible strain CBA / J female mice with a phosphate buffered saline solution containing freeze-dried pig thyroid powder on the 0th, 7th and 14th day after 8 weeks of age, and inject the EAT susceptible strain CBA / J female mice with a phosphate buffered saline solution containing lipopolysaccharide at a predetermined interval.

[0055] In this example, the phosphate buffered saline solution containing freeze-dried pig thyroid powder is a 100 μL phosphate buffered saline solution containing 1000 μg freeze-dried pig thyroid powder. The phosphate buffered saline solution containing lipopolysaccharide is a 100 μL phosphate buffered saline solution containing 100 μg lipopolysaccharide.

[0056] Specifically, the selected EAT susceptible strain CBA / J female mice (hereinafter referred to as mice) are injected with 1000 μg freeze-dried pig thyroid powder in 100 μL PBS (i.e. a sterile freeze-dried pig thyroid powder solution with a concentration of 10 ug / uL) via the tail vein on the 0th, 7th and 14th day after 8 weeks of age, and are injected with 100 μg LPS (lipopolysaccharide) in 100 μL PBS at an interval of 3 hours. That is, the mice are injected with 1000 μg freeze-dried pig thyroid powder in 100 μL PBS via the tail vein on the 0th day after 8 weeks of age, and are injected with 100 μg LPS (lipopolysaccharide) in 100 μL PBS at an interval of 3 hours; then the mice are injected with 1000 μg freeze-dried pig thyroid powder in 100 μL PBS via the tail vein on the 7th day after 8 weeks of age, and are injected with 100 μg LPS (lipopolysaccharide) in 100 μL PBS at an interval of 3 hours; then the mice are injected with 1000 μg freeze-dried pig thyroid powder in 100 μL PBS via the tail vein on the 14th day after 8 weeks of age, and are injected with 100 μg LPS (lipopolysaccharide) in 100 μL PBS at an interval of 3 hours.

[0057] S3: At the age of 12 weeks, the EAT susceptible strain CBA / J female mice are evaluated for modeling effect by thyroid pathological morphology and EAT score, serum TgAb concentration, serum TPOAb concentration and number of each subpopulation of spleen T lymphocytes.

[0058] Specifically, the mice are killed at 12 weeks of age, and the tolerance effect is measured by thyroid pathological morphology and EAT score, serum TgAb and TPOAb concentration, and spleen T lymphocyte in vitro stimulation proliferation level (S.I.).

[0059] Another embodiment of the present application also provides a Hashimoto's thyroiditis model, which is constructed by using the above construction method.

[0060] Another embodiment of the present application also provides an antigen for constructing a Hashimoto's thyroiditis model, which is a sterile porcine thyroid lyophilized powder solution with a concentration of 10 ug / uL, specifically a PBS solution of porcine thyroid lyophilized powder, wherein the concentration of the porcine thyroid lyophilized powder is 10 ug / uL. The active ingredients of the antigen are thyroglobulin and thyroid peroxidase. The antigen is an injection.

[0061] Specifically, the selected EAT susceptible strain CBA / J female mice (hereinafter referred to as mice) are injected with the antigen at 0, 7, and 14 days after 8 weeks of age, specifically 100 uL of PBS containing 1000 ug of porcine thyroid lyophilized powder, and 100 uL of PBS containing 100 ug of LPS (lipopolysaccharide) is injected at an interval of 3 hours. The porcine thyroid lyophilized powder can construct a Hashimoto's thyroiditis model by inducing the production of thyroid autoantibodies.

[0062] Embodiment Two

[0063] Based on the above embodiment, the present application provides a specific construction method of a Hashimoto's thyroiditis model, which comprises the following steps:

[0064] Step 1: Prepare porcine thyroid lyophilized powder, which can construct a Hashimoto's thyroiditis model by inducing the production of thyroid autoantibodies. The specific preparation process is described in Embodiment One, which is not repeated here.

[0065] Step 2: Select 8-week-old EAT susceptible strain CBA / J female mice, and inject the EAT susceptible strain CBA / J female mice with a phosphate buffered saline solution containing porcine thyroid lyophilized powder at 0, 7, and 14 days after 8 weeks of age, and inject a phosphate buffered saline solution containing lipopolysaccharide at a predetermined interval.

[0066] Please refer to Figure 3 , Figure 3is the experimental flow chart of CBA / J female mice using pig thyroid lyophilized powder to induce Hashimoto's thyroiditis provided by the embodiment of the present application. In this embodiment, 8-week-old EAT susceptible strain CBA / J female mice are randomly divided into a normal control group (NC group), a positive model group (pTg group) and a model group (pTLP group); for the model group (pTLP group) mice, 1000 μg of pig thyroid lyophilized powder is injected into the tail vein of the mice on the 0th day, the 7th day and the 14th day after reaching 8 weeks of age, and 100 μL of PBS containing 100 μg of LPS is injected into the tail vein of the mice at an interval of 3 hours; for the positive control group (pTg group) mice, 1000 μg of pig thyroid globulin (pTg) is injected into the tail vein of the mice on the 0th day, the 7th day and the 14th day after reaching 8 weeks of age, and 100 μL of PBS containing 100 μg of LPS is injected into the tail vein of the mice at an interval of 3 hours; for the normal control group (NC group) mice, 100 μL of PBS without pig thyroid lyophilized powder and without pig thyroid globulin is injected into the tail vein of the mice on the 0th day, the 7th day and the 14th day after reaching 8 weeks of age, and 100 μL of PBS containing 100 μg of LPS is injected into the tail vein of the mice at an interval of 3 hours.

[0067] Step 3: The mice were sacrificed when the EAT susceptible strain CBA / J female mice were 12 weeks old (on the 28th day), and the thyroid pathological morphology and EAT (experimental autoimmune thyroiditis) score, serum TgAb concentration, serum TPOAb concentration and the number of each subpopulation of spleen T lymphocytes were measured to evaluate the modeling effect.

[0068] Among them, the specific implementation operation of mouse thyroid tissue pathological examination and EAT model score is as follows:

[0069] (I) Mouse dissection and specimen preservation

[0070] (1) Anesthetize the mouse with 4% chloral hydrate, remove the unilateral eyeball with forceps to preserve the peripheral blood, and after standing at room temperature for 2 hours, centrifuge to collect the upper serum.

[0071] (2) The mouse was sacrificed by cervical dislocation method, taken out and fixed, the neck skin was cut open, the subcutaneous tissue, fascia and muscle layer were separated layer by layer, the trachea was exposed, the part of the trachea from the upper edge of the thyroid cartilage to the entrance of the thoracic cavity (where the thyroid gland is attached) was taken out, and then immersed in formalin and paraffin-embedded.

[0072] (3) Cut the abdominal skin, take out the spleen and divide it into two parts, one part is put into a small culture dish containing sterile 1640 solution to prepare a single cell suspension of the spleen, and the other part is stored for later use.

[0073] (ii) The company was sent to make thyroid paraffin sections, and HE staining was performed. The mouse thyroid sections after HE staining were observed under a microscope. Two doctors familiar with the pathological changes of mouse thyroiditis were referred to the previous mouse EAT model inflammation scoring standard. The average score was taken after independent scoring. If there was a serious disagreement, it would be discussed again and scored. The area of thyroid lymphocyte infiltration was calculated by Image J software. The scoring standard is shown in Table 1:

[0074] Table 1 Mouse EAT model thyroid pathological scoring standard

[0075]

[0076]

[0077] See Figure 4 , Figure 4 is the HE staining of the thyroid of each group of mice (200x, scale bar 100 pm) and the scatter plot of the pathological score of thyroid inflammation of each group of mice. The severity score of thyroiditis is scored as 0-4: 0, normal histology; 1: less than 10% of thyroid lymphocyte infiltration; 2, 10-30% lymphocyte infiltration; 3, 30-50% lymphocyte infiltration; 4, more than 50% lymphocyte infiltration. There is no lymphocyte infiltration in the thyroid tissue of the normal control group, as shown in Figure 4 (a). The pathological results of the pTg group of mice showed that the thyroid had less lymphocyte infiltration, accompanied by destruction and atrophy of the thyroid follicle, as shown in Figure 4 (b). The pathological results of the pTLP group of mice showed that the thyroid had obvious lymphocyte infiltration, accompanied by destruction and atrophy of the thyroid follicle, as shown in Figure 4 (c). The lymphocyte infiltration score of the pTLP group of mice was significantly higher than that of the normal control group of mice (Wilcoxon rank sum test P<0.0001, as shown in Figure 4 (d)). In addition, the thyroid inflammation score of the pTLP group can reach 3 points, which is higher than that of the pTg group, as shown in Figure 4 (d).

[0078] Further, the specific operation of detecting the level of serum thyroid autoantibody of the mouse is as follows:

[0079] (1) ELISA method was used to detect the relative concentration of serum TgAb.

[0080] (1) The specific steps of screening positive serum include the following steps:

[0081] (a) Take 10 mL of prepared coating buffer, add 100 μg of pTg protein (porcine thyroid globulin), prepare pTg coating solution with a concentration of 10 μg / mL, take 96-well enzyme-labeled plates, add 100 μL of pTg coating solution to each well, seal with sealing film and place in a 4°C refrigerator for 12 h;

[0082] (b) Take out the enzyme-labeled plates, discard the pTg coating solution in the wells, gently pat dry on absorbent paper, add 300 μL of prepared washing buffer to each well, gently shake the enzyme-labeled plates, stand for 1 min, discard the washing buffer in the wells, pat dry on absorbent paper, and repeat the washing for 2 more times;

[0083] (c) Add 200 μL of blocking solution to each well, seal with sealing film, stand for 12 h at 4°C, and repeat the washing for 3 times;

[0084] (d) Take the mouse serum samples that have been stably placed in a 4°C refrigerator for more than 6 h, mix with PBS solution at a ratio of 1:999, add 100 μL of the diluted serum sample to each well, set three duplicate wells for each mouse serum sample, seal and stand for 12 h at 4°C;

[0085] (e) Take out the enzyme-labeled plates, discard the serum sample in the wells, and wash for 3 times;

[0086] (f) Take HRP-labeled goat anti-mouse IgG (IgG-HRP), dilute with PBS at a ratio of 1:5000, add 100 μL of the diluted IgG-HRP to each well, gently shake the enzyme-labeled plates, seal with sealing film, and stand for 1 h at room temperature;

[0087] (g) Discard the liquid in the plates, wash for 3 times, add 100 μL of TMB color developing agent to each well, seal and avoid light for 5 min;

[0088] (h) Immediately add 50 μL of 2 Mol / L sulfuric acid to each well to stop the color development;

[0089] (i) Immediately use an enzyme-labeled instrument to detect and read the OD (optical density) value of each well at a wavelength of 450 nm;

[0090] (j) Take the average value of the three wells of each mouse serum sample as the OD value of the sample. The upper and lower limits of the normal value are defined as (mean ± 2 times the standard deviation) of the OD value of the control group mouse serum sample. If the OD value of the model group exceeds the upper limit, it is defined as positive. In this way, the mouse serum samples with positive TgAb are preliminarily screened out.

[0091] (2) Draw a standard curve of TgAb concentration, which specifically includes the following steps:

[0092] (a) Take 100 μL of each of the three mouse TgAb positive sera screened in the previous step, mix evenly;

[0093] (b) Using gradient dilution method, dilute the mixed serum to 1:300, 1:900, 1:2700, 1:8100, 1:24300, 1:72900, 1:218700 gradient concentrations, and their relative concentrations are defined as 218700, 72900, 24300, 8100, 2700, 900 and 300 in turn, draw the concentration standard curve;

[0094] (c) Repeat the experimental steps in step (1), detect the TgAb level of each group of mouse serum, set 3 duplicate holes. According to the standard curve drawn, calculate the relative concentration of TgAb in each hole, and take the average value of three holes as the relative concentration of TgAb of the sample.

[0095] (II) ELISA method for detecting serum TPOAb, specifically including the following steps:

[0096] (1) According to the reagent kit instructions, take out the enzyme labeled plate and stand at room temperature for 1 h. Set up blank holes, standard holes and sample holes, and handle them respectively: blank holes without reagents, standard holes with different concentrations of standard 50 μL, sample holes with mouse serum samples 50 μL which have been stabilized in 4℃ refrigerator for 6 h in advance;

[0097] (2) Except for the blank holes, 100 μL of HRP labeled antibody is added to each hole, and after sealing with sealing film, it is placed in a 37℃ constant temperature incubator for 1 h;

[0098] (3) Take out the enzyme labeled plate, discard the liquid in the hole, pat and drain on the water paper, add 350 μL of washing liquid to each hole, shake gently for 10 s, stand for 1 min, then discard the liquid and pat dry on the water paper, repeat the plate washing for 5 times;

[0099] (4) Add 50 μL of color developing substrate A+B to each hole, incubate at 37℃ in the dark for 15 min, then add 50 μL of stop solution to each hole;

[0100] (5) Immediately use the enzyme labeled instrument to detect the OD value of each hole at 450 nm, draw the standard concentration curve according to the OD value of each standard, and calculate the TPOAb concentration value of each serum sample.

[0101] Please refer to Figure 5 , Figure 5 are the columnar graphs of relative concentration of TgAb and TPOAb of serum of each group of mice, wherein, Figure 5 (a) is the columnar graph of relative concentration of TgAb of serum of each group of mice; Figure 5 (b) is the columnar graph of relative concentration of TPOAb of serum of each group of mice; Figure 5(c) is the relative concentration of TT4 in the serum of each group of mice. As shown in Figure 5 (a) As shown, compared with the control group, the levels of thyroid globulin antibodies in the peripheral blood of all mice immunized with porcine thyroid lyophilized powder (pTLP group) or porcine thyroid globulin (pTg group) were increased. As shown in Figure 5 (b) As shown, compared with the control group, the levels of thyroid peroxidase antibodies in the peripheral blood of mice in the pTg group and the pTLP group were also increased. As shown in Figure 5 (c) As shown, compared with the control group, there was no significant difference in the concentration of TT4 in the peripheral blood of mice in the pTLP group.

[0102] Further, the specific operation of in vitro culture and stimulation of proliferation of mouse spleen T lymphocytes is as follows:

[0103] The following operations are all carried out in a sterile environment.

[0104] (I) Preparation of spleen single cell suspension

[0105] (1) Place a sterile small culture dish with a diameter of 3 cm on the super-clean table, and put 1 mL of sterile 1640 solution in the dish. Take a sterilized 200-mesh filter and cut it into several square blocks with a size of 4 cm x 4 cm, and cover one of the blocks on the dish;

[0106] (2) Place one-half of the spleen on the filter, cut it with a sterile ophthalmic scissors, and gently grind the tissue with a sterile 5 mL syringe pillow to make most of the tissue leak under the filter into the 1640 solution, leaving only a small amount of white connective tissue on the filter;

[0107] (3) Discard the filter, and transfer the 1640 containing spleen cells in the small culture dish to a sterile 15 mL centrifuge tube, and then rinse the culture dish with 1640 solution, 1 mL each time, a total of 2 times, and transfer the rinsing liquid into the centrifuge tube;

[0108] (4) Centrifuge at 4°C, 500g, for 5 min, discard the supernatant, gently vortex, add 4 mL of red blood cell lysis solution, mix well by blowing, and stand at room temperature for 4 min;

[0109] (5) Add 10 mL of PBS solution for dilution and mix well, and stand at room temperature for 1 min;

[0110] (6) Centrifuge at 4°C, 500g, for 5 min, discard the supernatant, and observe the color of the cell sediment at the bottom of the tube. If there are still a lot of red blood cells, repeat the red cell lysis once;

[0111] (7) Add 2 mL of PBS solution to resuspend the cells, take 10 μL of the cell suspension and place it in a 1.5 mL EP tube containing 990 μL of PBS, vortex to mix, and count the total number of cells and the number of live cells using a disposable cell counting plate and an automatic cell counter.

[0112] (II) In vitro stimulation and proliferation of splenic T lymphocytes

[0113] (1) Take the single-cell suspension system containing 6 x 105 cells described above, centrifuge at 4°C and 500g for 5 min, discard the supernatant, resuspend the cells with 620 μL of T lymphocyte basic proliferation medium prepared in advance, and plant in a sterile 96-well cell culture plate, 200 μL / well, 3 wells per mouse;

[0114] (2) Repeat step (1) above, replacing the basic proliferation medium with a T lymphocyte stimulation and proliferation medium;

[0115] (3) Place the above two 96-well cell culture plates in a 37°C, 5% CO2 cell incubator for 72 h;

[0116] (4) Take out the cell culture plate, add 20 μL of CCK-8 to each well, avoid light, gently shake the plate, and continue to culture at 37°C, 5% CO2 for 3 h;

[0117] (5) Take out the 96-well plate and use a microplate reader to detect the OD value of each well at 450 nm;

[0118] (6) Calculate the stimulation index (S.I.) of splenic T lymphocytes of each mouse, where S.I. = 3-hole average of OD value of T lymphocyte stimulation and proliferation medium / 3-hole average of OD value of T lymphocyte basic proliferation medium.

[0119] See Figure 6 , Figure 6 are flow cytometry and bar graphs of the proportion of Th17 and Treg cells in the spleen of mice in each group, wherein Figure 6 (a) is a flow cytometry graph of the proportion of Th17 and Treg cells in the spleen of mice in each group; Figure 6 (b) is a bar graph of the proportion of Th17 and Treg cells in the spleen of mice in each group. Th17 is a subpopulation of CD4 lymphocytes expressing surface marker IL-17A. Treg cells (Tregs) are a subpopulation of CD4 lymphocytes expressing surface marker CD25 and nuclear transcription factor Foxp3.

[0120] To investigate whether these lymphocyte subsets play a role in the development of Tg and TPO antibodies and hypothyroidism in the pre-treated mice, the proportion of these lymphocyte subsets in the spleen of the relevant mice that died 4 weeks after the initial injection was measured. Flow analysis showed that the proportion of TH17 in the pTLP group was higher than that in the normal control group (p<0.05). The proportion of Treg cells had the opposite result, suggesting that the function of Treg cells is opposite to that of Th17 cells. Th17 cells are a pro-inflammatory cell subset that recruits neutrophils, promotes inflammation and tissue damage. While Treg cells have an antagonistic effect, playing a negative regulatory role in autoimmune diseases in terms of tolerance and immune response to self antigens. In the above experiment, the proportion of Th17 cells in the spleen of the pTLP group of mice was increased, the proportion of Treg cells was decreased, and the Th17 / Treg ratio was increased. It is suggested that porcine thyroid lyophilized powder can induce the occurrence of autoimmune thyroiditis by affecting the Th17 / Treg balance.

[0121] The present application provides a method for constructing a Hashimoto's thyroiditis model. After three consecutive tail vein injections of a phosphate buffered saline solution containing porcine thyroid lyophilized powder and a phosphate buffered saline solution containing lipopolysaccharide, the mice developed autoantibodies and there was obvious lymphoid infiltration in the thyroid tissue, indicating that the model was successfully constructed. Porcine thyroid lyophilized powder can induce the occurrence of Hashimoto's thyroiditis, and this modeling method provides a new approach to the pathogenesis of Hashimoto's thyroiditis. By detecting the proportion of Th17 (CD4+IL-17A+) and Treg (CD4+CD25+Foxp3+) cells in the mouse spleen, the present application confirms that they play an important role in the pathogenesis of autoimmune thyroiditis in mice. This finding provides a solid theoretical basis and experimental data to support future in-depth study of the immune tolerance mechanism of Hashimoto's thyroiditis.

[0122] In several embodiments provided by the present application, it should be understood that the disclosed devices and methods can be implemented in other manners. For example, the above-described device embodiments are merely illustrative. For example, the division of the modules is merely a logical function division. There can be another division manner for actual implementation. For example, a plurality of modules or components can be combined or integrated into another system, or some features can be ignored or not executed.

[0123] In addition, each function module in each embodiment of the present application can be integrated in a processing module, or each module can exist physically, or two or more modules can be integrated in one module. The above-mentioned integrated module can be realized in the form of hardware or in the form of hardware plus software function module.

[0124] The above is further detailed description of the present application in combination with specific preferred embodiments, and cannot be deemed as limitation of the specific implementation of the present application to these descriptions. For those skilled in the art to which the present application belongs, without departing from the concept of the present application, a number of simple deductions or substitutions can be made, and all should be deemed as falling within the protection scope of the present application.

Claims

1. A method for constructing a Hashimoto's thyroiditis model, characterized in that, include: S1: Preparation of freeze-dried porcine thyroid gland powder; including: S1.1: Separate thyroid tissue from the neck of a freshly slaughtered pig; S1.2: Remove the connective tissue from the thyroid tissue in an ice bath, and grind the thyroid tissue after removing the connective tissue using a grinder to obtain a thyroid extract; S1.3: The thyroid extract is frozen overnight in a refrigerator and placed in liquid nitrogen for a predetermined time to pretreat the thyroid extract; S1.4: The pretreated thyroid extract was freeze-dried to obtain porcine thyroid lyophilized powder and then frozen for storage; S2: Eight-week-old EAT-susceptible female mice of the CBA / J strain were selected and injected with a phosphate-buffered saline solution containing lyophilized porcine thyroid powder on days 0, 7, and 14 after eight weeks of age, and then injected with a phosphate-buffered saline solution containing lipopolysaccharide at predetermined intervals; wherein, the antigenic active components in the phosphate-buffered saline solution containing lyophilized porcine thyroid powder include thyroglobulin and thyroid peroxidase. S3: At 12 weeks of age, the modeling effect of the EAT-susceptible strain CBA / J female mice was assessed by thyroid pathological morphology and EAT score, serum TgAb concentration, serum TPOAb concentration, and the number of each subset of T lymphocytes in the spleen; among them, the average EAT score of the model mice was greater than 2; the proportion of Th17 cells and the proportion of Treg cells in the spleen of the model mice were increased. S1.1 includes: Thyroid tissue was separated from the neck of freshly slaughtered pigs, placed in a phosphate-buffered saline solution at 4°C, and transported to the laboratory. The phosphate-buffered saline solution had a pH of 7.0, a phosphate concentration of 15 mmol / L, and a sodium chloride concentration of 140 mmol / L. S1.2 includes: The connective tissue in the thyroid tissue was removed in an ice bath, and the thyroid tissue after removing the connective tissue was cut into thyroid tissue blocks of 0.1~0.2g. The thyroid tissue block was placed in phosphate-buffered saline and ground in a tissue grinder at a temperature of 4°C. Then it was centrifuged at 3000 rpm for 15 min, wherein the volume ratio of the phosphate-buffered saline to the thyroid tissue block was 10:

1. After centrifugation, the supernatant was collected, and the supernatant was a thyroid extract. Following S1, the following is also included: In use, the lyophilized porcine thyroid powder is dissolved in a phosphate buffer solution to obtain a 10 μg / μL phosphate buffer solution containing the lyophilized porcine thyroid powder. The phosphate buffered saline solution containing lyophilized porcine thyroid powder is a 100 μL phosphate buffered saline solution containing 1000 μg of lyophilized porcine thyroid powder. The phosphate buffered aqueous solution containing lipopolysaccharide is a 100 μL phosphate buffered aqueous solution containing 100 μg of lipopolysaccharide.

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