Hashimoto thyroiditis model, construction method thereof and antigen

Abstract

The invention discloses a hashimoto thyroiditis model, a construction method thereof and an antigen. The construction method comprises the following steps: preparing pig thyroid freeze-dried powder; the method comprises the following steps: selecting 8-week-old EAT susceptible strain CBAA / J female rats, respectively injecting a phosphate buffer saline solution containing pig thyroid freeze-dried powder on the 0th day, the 7th day and the 14th day after 8 weeks, and injecting a phosphate buffer saline solution containing lipopolysaccharide at preset time intervals; when the EAT susceptible strain CBAA / J female mouse is 12 weeks old, the modeling effect is measured according to the thyroid pathological morphology and EAT score, the serum TgAb concentration, the serum TPOAb concentration and the number of all subgroups of spleen T lymphocytes. After a mouse is subjected to continuous three-time caudal vein injection of a phosphate buffer saline solution containing the pig thyroid freeze-dried powder and lipopolysaccharide, autoantibodies are generated, and obvious lymphatic infiltration occurs in thyroid tissues, so that the pig thyroid freeze-dried powder can induce hashimoto thyroiditis, and a new thought is provided for pathogenesis of hashimoto thyroiditis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a Hashimoto's thyroiditis model and a construction method and antigen thereof. Background Art

[0002] Hashimoto's thyroiditis (HT), also known as chronic lymphocytic thyroiditis (CLT) or autoimmune thyroiditis (AIT), is the most common autoimmune disease, with a population incidence of 8 / 1000 to 46 / 1000. HT patients mainly present with diffuse enlargement of the thyroid gland, with a large number of lymphocytes infiltrating, extensive destruction and atrophy of the thyroid follicles, fibrosis of the lymphatic germinal centers, and gradual conversion of the patient's thyroid function to hypothyroidism or subclinical hypothyroidism. The serum thyroid peroxidase antibody (TPOAb) and / or thyroglobulin antibody (TgAb) levels are significantly increased. HT is the most common cause of clinical hypothyroidism, which can cause more than 90% of primary hypothyroidism. At the same time, the quality of life of HT patients is significantly lower than that of normal people, and their risk of cardiovascular and cerebrovascular diseases, metabolic syndrome, depression and thyroid cancer is also significantly increased.

[0003] At present, the pathogenesis of Hashimoto's thyroiditis is not fully understood. Therefore, it is very necessary to construct a stable and efficient Hashimoto's thyroiditis model. Summary of the invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides a Hashimoto's thyroiditis model and its construction method and antigen. The technical problem to be solved by the present invention is achieved by the following technical solutions:

[0005] The present invention provides a method for constructing a Hashimoto's thyroiditis model, comprising:

[0006] S1: Preparation of porcine thyroid freeze-dried powder;

[0007] S2: 8-week-old EAT-susceptible CBA / J female mice were selected and injected with phosphate-buffered saline solution containing porcine thyroid freeze-dried powder on days 0, 7, and 14 after 8 weeks of age, and phosphate-buffered saline solution containing lipopolysaccharide was injected at predetermined intervals;

[0008] S3: When the EAT-susceptible CBA / J female mice were 12 weeks old, the modeling effect was measured from the thyroid pathological morphology and EAT score, serum TgAb concentration, serum TPOAb concentration and the number of each T lymphocyte subset in the spleen.

[0009] In one embodiment of the present invention, the S1 includes:

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

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

[0012] S1.3: pre-treating the thyroid extract by freezing the thyroid extract in a refrigerator overnight and placing the thyroid extract in liquid nitrogen for a predetermined time;

[0013] S1.4: The pre-treated thyroid extract is freeze-dried to obtain porcine thyroid freeze-dried powder and stored frozen.

[0014] In one embodiment of the present invention, the S1.1 includes:

[0015] Thyroid tissue was isolated from the neck of a freshly slaughtered pig, placed in phosphate buffered saline at 4°C and transported to the laboratory, wherein the pH value of the phosphate buffered saline was 7.0, the concentration of phosphate in the phosphate buffered saline was 15 mMol / L, and the concentration of sodium chloride was 140 mMol / L.

[0016] In one embodiment of the present invention, the S1.2 includes:

[0017] Removing connective tissue from the thyroid tissue in an ice bath, and cutting the thyroid tissue after the connective tissue is removed into thyroid tissue blocks of 0.1 to 0.2 g;

[0018] Removing connective tissue from the thyroid tissue in an ice bath, and cutting the thyroid tissue after the connective tissue is removed into thyroid tissue blocks of 0.1 to 0.2 g;

[0019] The thyroid tissue block is placed in phosphate buffered saline and ground in a tissue grinder at a grinding temperature of 4° C., followed by centrifugation at a speed of 3000 rpm / min for 15 min, wherein the volume ratio of the phosphate buffered saline to the thyroid tissue block is 10:1;

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

[0021] In one embodiment of the present invention, after S1, the method further includes:

[0022] When in use, the porcine thyroid freeze-dried powder is dissolved in phosphate buffered saline solution to obtain a 10 ug / ul sterile porcine thyroid freeze-dried powder solution.

[0023] In one embodiment of the present invention, the phosphate-buffered saline solution containing porcine thyroid freeze-dried powder is 100 μL of phosphate-buffered saline solution containing 1000 μg of porcine thyroid freeze-dried powder.

[0024] In one embodiment of the present invention, 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 invention provides a Hashimoto's thyroiditis model, which is constructed using the construction method described in any one of the above embodiments.

[0026] Another aspect of the present invention provides an antigen for constructing a Hashimoto's thyroiditis model, wherein the antigen is a sterile porcine thyroid freeze-dried powder solution with a concentration of 10 ug / ul, and the active ingredients of the antigen are thyroglobulin and thyroid peroxidase.

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

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

[0029] 1. The present invention 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 freeze-dried powder and a phosphate-buffered saline solution containing lipopolysaccharide into female mice of the EAT susceptible strain CBA / J, autoantibodies were produced, and obvious lymphatic infiltration appeared in the thyroid tissue, indicating that the model was successfully constructed. Porcine thyroid freeze-dried powder can induce the occurrence of Hashimoto's thyroiditis, and this modeling method provides a new idea for the pathogenesis of Hashimoto's thyroiditis.

[0030] 2. The present invention confirmed the important role of Th17 (CD4+IL-17A+) and Treg (CD4+CD25+Foxp3+) cells in the pathogenesis of autoimmune thyroiditis in mice by detecting the cell ratios of Th17 (CD4+IL-17A+) and Treg (CD4+CD25+Foxp3+) in the spleen of mice. This discovery provides a relatively solid theoretical basis and experimental data support for future in-depth research on the immune tolerance mechanism of Hashimoto's thyroiditis.

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a flow chart of a method for constructing a Hashimoto's thyroiditis model provided by an embodiment of the present invention;

[0033] Figure 2 This is a flow chart of the preparation of a porcine thyroid freeze-dried powder provided in an embodiment of the present invention;

[0034] Figure 3 This is a flow chart of an experiment for inducing Hashimoto's thyroiditis in CBA / J female mice using porcine thyroid freeze-dried powder provided in an embodiment of the present invention;

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

[0036] Figure 5 is the bar graph of relative concentration of TgAb and TPOAb in the serum of each group of mice;

[0037] Figure 6 These are the flow cytometry and bar graphs showing the percentage of Th17 and Treg cells in the spleen of each group of mice. DETAILED DESCRIPTION

[0038] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the following is a detailed description of a Hashimoto's thyroiditis model and its construction method and antigen proposed according to the present invention in combination with the accompanying drawings and specific implementation methods.

[0039] The above and other technical contents, features and effects of the present invention are clearly presented in the following detailed description of the specific implementation modes in conjunction with the accompanying drawings. Through the description of the specific implementation modes, the technical means and effects adopted by the present invention to achieve the predetermined purpose can be more deeply and specifically understood. However, the attached drawings are only for reference and explanation purposes and are not used to limit the technical solutions of the present invention.

[0040] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants are intended to cover non-exclusive inclusion, so that an article or device including a series of elements includes not only those elements, but also other elements that are not explicitly listed. In the absence of more restrictions, the elements defined by the statement "including one..." do not exclude the existence of other identical elements in the article or device including the elements.

[0041] Embodiment 1

[0042] See also Figure 1 , Figure 11 is a flow chart of a method for constructing a Hashimoto's thyroiditis model provided by an embodiment of the present invention. The construction method comprises the following steps:

[0043] S1: Prepare porcine thyroid freeze-dried powder.

[0044] like Figure 2 As shown, step S1 of this embodiment specifically includes:

[0045] S1.1: Thyroid tissue was isolated from the neck of freshly slaughtered pigs.

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

[0047] S1.2: The connective tissue in the thyroid tissue is removed in an ice bath, and the thyroid tissue after the connective tissue is removed is ground using a grinder to obtain a thyroid extract.

[0048] Specifically, the connective tissues such as fat and capsule in the thyroid tissue are removed in an ice bath, and the thyroid tissue after the connective tissue is removed is cut into 0.1-0.2g thyroid tissue blocks; the thyroid tissue blocks are placed in 10 tissue 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, and the grinding is twice, each time for 60 seconds, the interval between the two grindings is 15 seconds, and the grinding power is 60Hz. The model of the low-temperature tissue grinder used in this embodiment is Jingxin JXFSTPRP-III. Subsequently, the supernatant is recovered after centrifugation at a speed of 3000rpm / min for 15min in a centrifuge, and the supernatant is a thyroid extract.

[0049] S1.3: Pre-treat the thyroid extract by freezing it in a refrigerator overnight and placing it in liquid nitrogen for a predetermined time.

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

[0051] S1.4: The pre-treated thyroid extract is freeze-dried to obtain porcine thyroid freeze-dried powder and stored frozen.

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

[0053] Furthermore, when used, the porcine thyroid freeze-dried powder is dissolved in phosphate buffered saline solution to form a 10 ug / ul sterile porcine thyroid freeze-dried powder solution.

[0054] S2: 8-week-old EAT-susceptible CBA / J female mice were selected as the model group (pTLP group). On the 0th, 7th and 14th days after 8 weeks of age, the EAT-susceptible CBA / J female mice were injected with phosphate-buffered saline solution containing porcine thyroid freeze-dried powder, and phosphate-buffered saline solution containing lipopolysaccharide was injected at predetermined intervals.

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

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

[0057] S3: When the EAT-susceptible CBA / J female mice were 12 weeks old, the modeling effect was measured by thyroid pathological morphology and EAT score, serum TgAb concentration, serum TPOAb concentration and the number of each T lymphocyte subsets in the spleen.

[0058] Specifically, the mice were killed at 12 weeks of age, and the tolerance effect was measured based on the thyroid pathological morphology and EAT score, serum TgAb, TPOAb concentrations, and in vitro stimulated proliferation level (SI) of splenic T lymphocytes.

[0059] Another embodiment of the present invention further provides a Hashimoto's thyroiditis model, which is constructed using the above-mentioned construction method.

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

[0061] Specifically, the antigen was injected into the tail vein of the selected EAT susceptible strain CBA / J female mice (hereinafter referred to as mice) on days 0, 7, and 14 after they were 8 weeks old. Specifically, 100 μL PBS containing 1000 μg of porcine thyroid freeze-dried powder was injected, and 100 μL PBS containing 100 μg of LPS (lipopolysaccharide) was injected at intervals of 3 hours. Porcine thyroid freeze-dried powder can induce the production of thyroid autoantibodies to construct a Hashimoto's thyroiditis model.

[0062] Embodiment 2

[0063] Based on the above embodiments, the present invention provides a specific method for constructing a Hashimoto's thyroiditis model, which comprises:

[0064] Step 1: Prepare porcine thyroid freeze-dried powder, which induces the production of thyroid autoantibodies to construct a Hashimoto's thyroiditis model. The specific preparation process is shown in Example 1 and will not be described again here.

[0065] Step 2: Select 8-week-old EAT-susceptible strain CBA / J female mice, and inject them with phosphate-buffered saline solution containing freeze-dried porcine thyroid powder on day 0, day 7 and day 14 after 8 weeks of age, and inject them with phosphate-buffered saline solution containing lipopolysaccharide at predetermined intervals.

[0066] See also Figure 3 , Figure 3This is an experimental flow chart of inducing Hashimoto's thyroiditis in CBA / J female mice using porcine thyroid freeze-dried powder provided in an embodiment of the present invention. In this embodiment, 8-week-old EAT susceptible strain CBA / J female mice were randomly divided into a normal control group (NC group), a positive model group (pTg group) and a model group (pTLP group); for the mice in the model group (pTLP group), on the 0th day, the 7th day and the 14th day after they were 8 weeks old, 100 μL PBS containing 1000 μg porcine thyroid freeze-dried powder was injected into the tail vein, and 100 μL PBS containing 100 μg LPS was injected at an interval of 3 hours; for the mice in the positive control group (pTg group), on the 0th day, the 7th day and the 14th day after they were 8 weeks old, 100 μL PBS containing 1000 μg porcine thyroglobulin (pTg) was injected into the tail vein, and 100 μL PBS containing 100 μg LPS was injected at an interval of 3 hours; for the mice in the normal control group (NC group), on the 0th day, the 7th day and the 14th day after they were 8 weeks old, 100 μL PBS containing no porcine thyroid freeze-dried powder and no porcine thyroglobulin was injected into the tail vein. PBS, 100 μL PBS containing 100 μg LPS was injected at 3 h intervals.

[0067] Step 3: The EAT-susceptible CBA / J female mice were killed at 12 weeks of age (day 28), and the modeling effect was measured based on the thyroid pathological morphology and EAT (experimental autoimmune thyroiditis) score, serum TgAb concentration, serum TPOAb concentration, and the number of each T lymphocyte subset in the spleen.

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

[0069] (I) Mouse anatomical processing and specimen collection

[0070] (1) Mice were anesthetized with 4% chloral hydrate, and peripheral blood was collected by removing one eyeball with forceps. After standing at room temperature for 2 hours, the upper serum was collected by centrifugation.

[0071] (2) Mice were killed by cervical vertebral dissection, removed and fixed, the neck skin was cut open, the subcutaneous tissue, fascia and muscle layer were separated layer by layer, the trachea was exposed, and the trachea section from the upper edge of the thyroid cartilage to the thoracic inlet (where the thyroid gland is attached) was taken, immersed in formalin, and embedded in paraffin.

[0072] (3) Cut the abdominal skin open, remove the spleen and divide it into two equal parts. Place one part in a small culture dish containing sterile 1640 solution to prepare a spleen single cell suspension, and freeze the other part for later use.

[0073] (II) The samples were sent to the company for paraffin sectioning of the thyroid gland and HE staining. The HE-stained mouse thyroid sections were observed under a microscope. Two physicians familiar with the pathological changes of mouse thyroiditis used the previous mouse EAT model inflammation score criteria to score the samples independently and average the scores. If there were serious disagreements, the scores were discussed and re-scored. The percentage of thyroid lymphocyte infiltration area was calculated using Image J software. The scoring criteria are shown in Table 1:

[0074] Table 1 Scoring criteria for thyroid pathology in the mouse EAT model

[0075]

[0076]

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

[0078] Furthermore, the specific operation of detecting the level of thyroid autoantibodies in mouse serum is as follows:

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

[0080] (1) Initial screening of positive serum, specifically including the following steps:

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

[0082] (b) Take out the ELISA plate, pour out the pTg coating solution in the wells, gently pat dry on absorbent paper, add 300 μL of the previously prepared washing buffer to each well, shake the ELISA plate gently, let it stand for 1 min, pour out the washing buffer in the wells, pat dry on absorbent paper, and repeat the washing twice;

[0083] (c) Add 200 μL of blocking solution to each well, seal the plate with sealing film, and incubate at 4°C for 12 h. Take out the ELISA plate and wash it three times.

[0084] (d) Take a mouse serum sample that has been stabilized in a 4°C refrigerator for more than 6 hours, mix and dilute it with PBS solution at a ratio of 1:999, add 100 μL of the diluted serum sample to each well, set three replicate wells for each mouse serum sample, seal them and let them stand at 4°C for 12 hours;

[0085] (e) Take out the ELISA plate, discard the serum samples in the wells, and wash three times;

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

[0087] (g) Pour off the liquid in the plate, wash three times, add 100 μL of TMB colorimetric reagent to each well, seal and keep away from light for 5 min;

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

[0089] (i) Immediately use a microplate reader to read the OD (optical density) value of each well at a wavelength of 450 nm;

[0090] (j) The average value of three wells of each mouse serum sample tested was defined as the OD value of the sample. The upper and lower limits of the normal value were defined by the OD value (mean ± 2 times standard deviation) of the mouse serum sample in the control group. If the OD value of the model group exceeded the upper limit, it was defined as positive, so as to preliminarily screen out the mouse serum samples with positive TgAb.

[0091] (2) Drawing a TgAb concentration standard curve, specifically comprising the following steps:

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

[0093] (b) using a gradient dilution method, the mixed serum was diluted to a gradient concentration of 1:300, 1:900, 1:2700, 1:8100, 1:24300, 1:72900, and 1:218700, and its relative concentrations were defined as 218700, 72900, 24300, 8100, 2700, 900, and 300, and a concentration standard curve was drawn;

[0094] (c) Repeat the experimental steps in step (1) to detect the serum TgAb level of each group of mice, set up 3 replicate wells, calculate the relative concentration of TgAb in each well according to the drawn standard curve, and take the average value of the three wells as the relative concentration of TgAb in the sample.

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

[0096] (1) According to the instructions of the kit, take out the ELISA plate and let it stand at room temperature for 1 hour. Set up blank wells, standard wells and sample wells for treatment: no reagent is added to the blank wells, 50 μL of standards of different concentrations are added to the standard wells, and 50 μL of mouse serum samples that have been stabilized in a 4°C refrigerator for 6 hours are added to the sample wells;

[0097] (2) Except for the blank well, add 100 μL of HRP-labeled antibody to each well, seal with a sealing film, and incubate in a 37°C incubator for 1 h;

[0098] (3) Take out the ELISA plate, discard the liquid in the wells, tap and drain on absorbent paper, add 350 μL of washing solution to each well, shake gently for 10 seconds, let it stand for 1 minute, discard the liquid, tap dry on absorbent paper, and repeat washing the plate 5 times;

[0099] (4) Add 50 μL of chromogenic substrate A+B to each well, incubate at 37°C in the dark for 15 min, and then add 50 μL of stop solution to each well;

[0100] (5) Immediately use an ELISA instrument to detect the OD value of each well at 450 nm, draw a standard concentration curve based on the OD value of each standard, and calculate the TPOAb concentration value of each serum sample.

[0101] See also Figure 5 , Figure 5 The relative concentration bar graphs of TgAb and TPOAb in the serum of each group of mice are shown in Figure 2. Figure 5 (a) is a bar graph of the relative concentration of serum TgAb in each group of mice; Figure 5 (b) is a bar graph of the relative concentration of serum TPOAb in each group of mice; Figure 5(c) is a bar graph of the relative concentration of TT4 in the serum of each group of mice. Figure 5 As shown in (a), compared with the control group (control), the levels of thyroglobulin antibodies in the peripheral blood of all mice immunized with porcine thyroid freeze-dried powder (pTLP group) or porcine thyroglobulin (pTg group) were increased. Figure 5 As shown in (b), compared with the control group, the levels of thyroid peroxidase antibodies in the peripheral blood of mice in the pTg and pTLP groups were also increased. Figure 5 As shown in (c), there was no significant difference in the concentration of TT4 in the peripheral blood of mice in the pTLP group compared with that in the normal control group.

[0102] Furthermore, the specific operations of in vitro culture and proliferation stimulation of mouse spleen T lymphocytes are as follows:

[0103] The following operations were all performed under a sterile environment.

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

[0105] (1) Take a 3 cm diameter sterile small culture dish and place it on the clean bench. The dish contains 1 mL of sterile 1640 solution. Take a sterilized 200 mesh filter and cut it into several square pieces of 4 cm × 4 cm. Take one piece and cover it on the dish.

[0106] (2) Place half of the spleen on the filter, cut it into pieces with sterile ophthalmic scissors, and gently grind the tissue with the pillow of a sterile 5 mL syringe, so that most of the tissue leaks under the filter and enters 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. Rinse the culture dish twice with 1 mL of 1640 solution each time, and transfer the rinse solution to the centrifuge tube.

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

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

[0110] (6) Centrifuge at 4°C, 500 g for 5 min, discard the supernatant, and observe the color of the cell pellet at the bottom of the tube. If it still contains a large number of red blood cells, repeat the red blood cell splitting 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 well. Use a disposable cell counting plate with an automatic cell counter to count the total number of cells and the number of live cells.

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

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

[0114] (2) repeating the above step (1), replacing the basal proliferation medium with T lymphocyte stimulation proliferation medium;

[0115] (3) placing the two 96-well cell culture plates in a 37° C., 5% CO 2 cell culture incubator and culturing for 72 h;

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

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

[0118] (6) Calculate the stimulation index (SI) of spleen T lymphocytes of each mouse, where SI = the average of the OD values ​​of the T lymphocyte stimulation medium for three wells / the average of the OD values ​​of the T lymphocyte basal medium for three wells.

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

[0120] In order to study whether these lymphocyte subsets play a role in pretreatment mice that cause Tg and TPO antibodies and hypothyroidism, this example measures the proportion of these lymphocyte subsets in the spleen of related mice that died 4 weeks after the initial injection. 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 Treg cell ratio had a completely opposite result, suggesting that the function of Treg cells is opposite to that of Th17 cells. Th17 cells are proinflammatory cell subsets that recruit neutrophils to promote inflammation and tissue damage. Treg cells have antagonistic effects and play a negative regulatory role in tolerance to self-antigens and immune responses in autoimmune diseases. In the above experiment, the proportion of Th17 cells in the spleen of mice in the pTLP group increased, the proportion of Treg cells decreased, and the Th17 / Treg ratio increased. It is suggested that pig thyroid freeze-dried powder may induce the occurrence of autoimmune thyroiditis by affecting the Th17 / Treg balance.

[0121] The present invention 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 freeze-dried powder and a phosphate-buffered saline solution containing lipopolysaccharide, mice produced autoantibodies, and obvious lymphatic infiltration appeared in the thyroid tissue, indicating that the model was successfully constructed. Porcine thyroid freeze-dried powder can induce the occurrence of Hashimoto's thyroiditis, and this modeling method provides a new idea for the pathogenesis of Hashimoto's thyroiditis. The present invention confirms that the cell ratios of Th17 (CD4+IL-17A+) and Treg (CD4+CD25+Foxp3+) in the spleen of mice play an important role in the onset of autoimmune thyroiditis in mice by detecting them. This discovery provides a relatively solid theoretical basis and experimental data as support for in-depth research on the immune tolerance mechanism of Hashimoto's thyroiditis in the future.

[0122] In the several embodiments provided by the present invention, it should be understood that the apparatus and method disclosed by the present invention can be implemented in other ways. For example, the apparatus embodiments described above are only illustrative, for example, the division of the modules is only a logical function division, and there may be other division methods in actual implementation, for example, multiple modules or components can be combined or integrated into another system, or some features can be ignored or not executed.

[0123] In addition, each functional module in each embodiment of the present invention may be integrated into one processing module, or each module may exist physically separately, or two or more modules may be integrated into one module. The above-mentioned integrated module may be implemented in the form of hardware or in the form of hardware plus software functional modules.

[0124] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A method for constructing a Hashimoto's thyroiditis model, characterized in that: include: S1: Preparation of porcine thyroid freeze-dried powder; S2: 8-week-old EAT-susceptible CBA / J female mice were selected and injected with phosphate-buffered saline solution containing porcine thyroid freeze-dried powder on days 0, 7, and 14 after 8 weeks of age, and phosphate-buffered saline solution containing lipopolysaccharide was injected at predetermined intervals; S3: When the EAT-susceptible CBA / J female mice were 12 weeks old, the modeling effect was measured from the thyroid pathological morphology and EAT score, serum TgAb concentration, serum TPOAb concentration and the number of each T lymphocyte subset in the spleen.

2. The method for constructing a Hashimoto's thyroiditis model according to claim 1, characterized in that: The S1 includes: S1.1: Isolate thyroid tissue from the neck of freshly slaughtered pigs; S1.2: removing connective tissue from the thyroid tissue in an ice bath, and grinding the thyroid tissue after the connective tissue is removed using a grinder to obtain a thyroid extract; S1.3: pre-treating the thyroid extract by freezing the thyroid extract in a refrigerator overnight and placing the thyroid extract in liquid nitrogen for a predetermined time; S1.4: The pre-treated thyroid extract is freeze-dried to obtain porcine thyroid freeze-dried powder and stored frozen.

3. The method for constructing a Hashimoto's thyroiditis model according to claim 2, characterized in that: The S1.1 includes: Thyroid tissue was isolated from the neck of a freshly slaughtered pig, placed in phosphate buffered saline at 4°C and transported to the laboratory, wherein the pH value of the phosphate buffered saline was 7.0, the concentration of phosphate in the phosphate buffered saline was 15 mMol / L, and the concentration of sodium chloride was 140 mMol / L.

4. The method for constructing a Hashimoto's thyroiditis model according to claim 2, characterized in that: The S1.2 includes: Removing connective tissue from the thyroid tissue in an ice bath, and cutting the thyroid tissue after the connective tissue is removed into thyroid tissue blocks of 0.1 to 0.2 g; The thyroid tissue block is placed in phosphate buffered saline and ground in a tissue grinder at a grinding temperature of 4° C., followed by centrifugation at a speed of 3000 rpm / min for 15 min, wherein the volume ratio of the phosphate buffered saline to the thyroid tissue block is 10:1; After centrifugation, the supernatant is recovered, and the supernatant is the thyroid extract.

5. The method for constructing a Hashimoto's thyroiditis model according to claim 1, characterized in that: After S1, the following is also included: When in use, the porcine thyroid freeze-dried powder is dissolved in phosphate buffered saline solution to obtain a 10 ug / uL sterile porcine thyroid freeze-dried powder solution.

6. The method for constructing a Hashimoto's thyroiditis model according to claim 1, characterized in that: The phosphate buffered saline solution containing the porcine thyroid freeze-dried powder is 100 μL of phosphate buffered saline solution containing 1000 μg of porcine thyroid freeze-dried powder.

7. The method for constructing a Hashimoto's thyroiditis model according to claim 1, characterized in that: The phosphate buffered saline solution containing lipopolysaccharide is 100 μL of phosphate buffered saline solution containing 100 μg of lipopolysaccharide.

8. A Hashimoto's thyroiditis model, characterized in that: The method is constructed using any one of claims 1 to 6.

9. An antigen for constructing the Hashimoto's thyroiditis model according to claim 7, characterized in that: The antigen is a sterile porcine thyroid freeze-dried powder solution with a concentration of 10ug / ul, and the active components of the antigen are thyroglobulin and thyroid peroxidase.

10. The antigen according to claim 9, characterized in that The antigen is in the form of an injection.

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