Medicine for preventing hashimoto thyroiditis as well as preparation method and application thereof

Abstract

The invention discloses a medicine for preventing hashimoto thyroiditis as well as a preparation method and application thereof, the medicine is a 10 [mu] g / [mu] l sterile pig thyroid freeze-dried powder solution, and the active ingredients of the medicine are thyroglobulin and thyroid peroxidase. The preparation method comprises the following steps: separating thyroid tissues from necks of just slaughtered pigs; removing connective tissues in the thyroid tissues in an ice bath, and grinding the thyroid tissues from which the connective tissues are removed by using a grinding machine to obtain a thyroid extract; freezing the thyroid extract overnight and placing the thyroid extract in liquid nitrogen for a preset time so as to pre-treat the thyroid extract; and carrying out freeze-drying treatment on the pretreated thyroid extract to obtain pig thyroid freeze-dried powder, and carrying out cryopreservation on the pig thyroid freeze-dried powder. Immune tolerance can be induced by oral administration or subcutaneous injection of the pig thyroid freeze-dried powder prepared by the invention for a newborn CBA / J mouse, so that autoimmune thyroiditis after adult can be prevented.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a medicine for preventing Hashimoto's thyroiditis, and a preparation method and application 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 thyroid enlargement, with a large number of lymphocytes infiltrating, extensive destruction and atrophy of thyroid follicles, fibrosis of lymphoid germinal centers, and gradual conversion of thyroid function to hypothyroidism or subclinical hypothyroidism. 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] There is currently no effective drug for the treatment of Hashimoto's thyroiditis. Therefore, it is very necessary to provide a drug that can effectively induce tolerance to Hashimoto's thyroiditis in newborn mice. Summary of the invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides a drug for preventing Hashimoto's thyroiditis and its preparation method and application. The technical problem to be solved by the present invention is achieved by the following technical solution:

[0005] One aspect of the present invention provides a method for preparing a drug for preventing Hashimoto's thyroiditis, comprising:

[0006] S1: Thyroid tissue was isolated from the neck of freshly slaughtered pigs;

[0007] S2: removing connective tissue from the thyroid tissue in an ice bath, grinding the thyroid tissue after the connective tissue is removed using a grinder, and recovering a supernatant, wherein the supernatant is a thyroid extract;

[0008] S3: pre-treating the thyroid extract by freezing the thyroid extract overnight and placing the thyroid extract in liquid nitrogen for a predetermined time;

[0009] S4: freeze-dry the pre-treated thyroid extract to obtain pig thyroid freeze-dried powder and store it in a frozen state.

[0010] In one embodiment of the present invention, the S1 includes:

[0011] 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; the pH value of the phosphate buffered saline was 7.0, wherein the concentration of phosphate was 15 mMol / L and the concentration of NaCl was 140 mMol / L.

[0012] In one embodiment of the present invention, the S2 includes:

[0013] 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;

[0014] 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;

[0015] After centrifugation, the supernatant is recovered, and the supernatant is the thyroid extract.

[0016] In one embodiment of the present invention, S3 includes:

[0017] The thyroid extract was placed in a -80°C freezer overnight, and then placed in liquid nitrogen for at least 1 hour to pre-treat the thyroid extract.

[0018] In one embodiment of the present invention, the preparation method further comprises: when in use, dissolving the porcine thyroid freeze-dried powder into a 10 ug / ul sterile porcine thyroid freeze-dried powder solution using a phosphate buffered saline solution.

[0019] Another aspect of the present invention provides a drug for preventing Hashimoto's thyroiditis, which is prepared by the preparation method described in any one of the above embodiments, wherein the drug is a 10ug / uL sterile porcine thyroid lyophilized powder solution, and the active ingredients of the drug are thyroglobulin and thyroid peroxidase.

[0020] In one embodiment of the present invention, the drug is applied within 24 hours of the neonatal period, and is applied orally, subcutaneously, or intramuscularly.

[0021] Another aspect of the present invention provides use of a drug for preventing Hashimoto's thyroiditis in inducing tolerance in newborn mice.

[0022] In one embodiment of the present invention, the application includes:

[0023] Select EAT susceptible strain CBA / J newborn mice, and orally, subcutaneously or intramuscularly inject the mice with a dose of 60ug / g body weight and a concentration of 10ug / uL of sterile porcine thyroid freeze-dried powder solution within 24 hours after birth.

[0024] In one embodiment of the present invention, the application includes:

[0025] On days 0, 7, and 14 after the mice reached 8 weeks of age, 100 μL of phosphate-buffered saline solution containing 1000 μg of porcine thyroid freeze-dried powder was injected into the tail vein, and 100 μL of phosphate-buffered saline solution containing 100 μg of lipopolysaccharide was injected at intervals of 3 h.

[0026] Thyroid pathological morphology, EAT score, serum TgAb, TPOAb concentrations and in vitro stimulated proliferation levels of splenic T lymphocytes were obtained when the mice were 12 weeks old to measure the tolerance effect.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The present invention provides a drug for preventing Hashimoto's thyroiditis, and its preparation method and application. The drug is a 10ug / ul sterile porcine thyroid lyophilized powder solution, and the active ingredients of the drug are thyroglobulin and thyroid peroxidase. EAT-susceptible strain CBA / J newborn mice can be induced to induce immune tolerance by oral or subcutaneous injection of porcine thyroid lyophilized powder to prevent the occurrence of autoimmune thyroiditis in adulthood. Neonatal tolerance can prevent the occurrence of Hashimoto's thyroiditis. This immunoprevention method has high clinical practicality and operability, and provides a new direction for the immunoprevention of Hashimoto's thyroiditis. It provides a powerful explanation for the selection of antigen dosage, antigen administration method, and time window in which the host is prone to neonatal tolerance in future clinical work to prevent Hashimoto's thyroiditis through neonatal tolerance.

[0029] 2. The present invention detects the cell ratios of Th17 (CD4+IL-17A+) and Treg (CD4+CD25+Foxp3+) in the spleen of mice, confirming that oral administration and subcutaneous injection of pig thyroid freeze-dried powder in the neonatal period to induce immune tolerance has a preventive effect on the onset of autoimmune thyroiditis in adult mice, and can increase the number of Treg cells in the spleen of mice, reduce the number of Th17 cells, and reshape the balance of Th17 / Treg cells. This discovery provides a relatively solid theoretical basis and experimental data as support for future in-depth studies on the immune tolerance mechanism of Hashimoto's thyroiditis.

[0030] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a flow chart of a method for preparing a drug for preventing Hashimoto's thyroiditis provided by an embodiment of the present invention;

[0032] Figure 2 This is an experimental flow chart of inducing immune tolerance in newborn CBA / J female mice by oral administration, subcutaneous injection or intramuscular injection of porcine thyroid freeze-dried powder provided in an embodiment of the present invention;

[0033] Figure 3 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;

[0034] Figure 4 is the bar graph of relative concentration of TgAb and TPOAb in the serum of each group of mice;

[0035] Figure 5 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

[0036] 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 drug for preventing Hashimoto's thyroiditis and its preparation method and application proposed in accordance with the present invention in combination with the accompanying drawings and specific implementation methods.

[0037] 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.

[0038] 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.

[0039] Embodiment 1

[0040] See also Figure 1 , Figure 1 1 is a flow chart of a method for preparing a drug for preventing Hashimoto's thyroiditis provided by an embodiment of the present invention. The preparation method comprises:

[0041] S1: Thyroid tissue was isolated from the neck of freshly slaughtered pigs.

[0042] 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.

[0043] S2: 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.

[0044] 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 (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, the grinding power is 60Hz, and the model of the low-temperature tissue grinder used in this embodiment is Jingxin JXFSTPRP-III. Subsequently, the centrifuge is centrifuged at a speed of 3000rpm / min for 15min, and the supernatant is recovered after the centrifugation, and the supernatant is the thyroid extract.

[0045] S3: Pre-treat the thyroid extract by freezing it in a refrigerator overnight and placing it in liquid nitrogen for a predetermined time.

[0046] 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.

[0047] S4: freeze-dry the pre-treated thyroid extract to obtain pig thyroid freeze-dried powder and store it in a frozen state.

[0048] 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.

[0049] Furthermore, when in use, 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.

[0050] Embodiment 2

[0051] On the basis of Example 1, this example provides a drug for preventing Hashimoto's thyroiditis, which is a 10ug / ul sterile porcine thyroid lyophilized powder solution, wherein the solvent is a phosphate buffered saline solution, and the active ingredients of the drug are thyroglobulin and thyroid peroxidase.

[0052] Furthermore, the drug for preventing Hashimoto's thyroiditis is applied within 24 hours of the newborn period, and the application method is oral, subcutaneous injection and intramuscular injection to ensure the best preventive effect. Preferably, the dosage is 60ug / g body weight, that is, 60ug is administered for every 1g of body weight.

[0053] Embodiment 3

[0054] Based on the above embodiments, this embodiment provides a use of a drug for preventing Hashimoto's thyroiditis in inducing tolerance in newborn mice, comprising:

[0055] Select EAT susceptible strain CBA / J newborn mice, and orally, subcutaneously or intramuscularly inject the mice with a dose of 60ug / g body weight and a concentration of 10ug / uL of sterile porcine thyroid freeze-dried powder solution within 24 hours after birth.

[0056] The application process also includes:

[0057] On days 0, 7, and 14 after the mice were 8 weeks old, 100 μL of phosphate-buffered saline solution containing 1000 μg of porcine thyroid freeze-dried powder was injected into the tail vein, and 100 μL of phosphate-buffered saline solution containing 100 μg of lipopolysaccharide (LPS) was injected every 3 hours; the thyroid pathological morphology, EAT score, serum TgAb, TPOAb concentrations, and in vitro stimulated proliferation of spleen T lymphocytes were obtained at 12 weeks of age to measure the tolerance effect. The specific application principle is: porcine thyroid freeze-dried powder regulates the balance of Th17 / Treg cells by inducing neo-tolerance and preventing the occurrence of Hashimoto's thyroiditis.

[0058] Embodiment 4

[0059] Based on the above embodiments, this embodiment provides a method for constructing a Hashimoto's thyroiditis model based on the EAT susceptible strain CBA / J mice, comprising the following steps:

[0060] Step 1: Select EAT susceptible strain CBA / J newborn mice as the prevention experimental group.

[0061] Step 2: Within 24 hours after birth, mice in the prevention experimental group were given oral, subcutaneous or intramuscular injections of a sterile thyroid freeze-dried powder solution with a dose of 60ug / g body weight and a concentration of 10ug / uL.

[0062] Specifically, see Figure 2 , Figure 2 This is an experimental flow chart of inducing immune tolerance by oral, subcutaneous or intramuscular injection of porcine thyroid freeze-dried powder in newborn CBA / J female mice provided by an embodiment of the present invention. The method of administering porcine thyroid freeze-dried powder to newborn CBA / J mice is as follows:

[0063] Step 2.1: Dissolve the porcine thyroid freeze-dried powder in sterile PBS to prepare a sterile porcine thyroid freeze-dried powder solution with a concentration of 10ug / uL. Note that this should be done 20 minutes in advance and equilibrated to room temperature. When operating, be sure to touch the bedding in the cage before touching the mouse to avoid different odors irritating the mother mouse.

[0064] Step 2.2: First weigh the mice (i.e., EAT susceptible strain CBA / J newborn mice). The control group (control group) and the modeling group (pTLP group) were orally administered with an equal volume of sterile PBS. The tolerance group (including PO (oral) group, SC (subcutaneous injection) group, and IM (muscular injection) group) was orally, subcutaneously, or intramuscularly injected with a sterile thyroid freeze-dried powder solution of 60ug / g body weight and a concentration of 10ug / ul within 24 hours after birth.

[0065] The specific operation of oral administration (PO group) was to use a sterile 10μL pipette to gently inject into the pharynx from one corner of the mouth of the newborn mouse to stimulate the upper palate, induce the mouse's swallowing reflex, ensure the efficiency of oral administration, and prevent accidental inhalation into the trachea causing the mouse to choke and cough; the specific operation of subcutaneous injection (SC group) was to gently lift the skin of the back of the mouse's neck, and slowly inject the corresponding volume of sterile porcine thyroid freeze-dried powder solution with an insulin syringe; the specific operation of intramuscular injection (IM group) was to gently fix the mouse, straighten the mouse's left thigh, and slowly inject the corresponding volume of sterile porcine thyroid freeze-dried powder solution from the inner muscle.

[0066] (3) After the operation is completed, the newborn mice are returned to the mouse nest and the mother mouse continues to breastfeed them. After one week, the gender of the mice is determined again and recorded. At three weeks of age, the mice are weaned and raised until they are 8 weeks old.

[0067] Step 3: One year later, the prevention experimental group used porcine thyroid freeze-dried powder and LPS to induce immune modeling. The tolerance effect was measured from the thyroid pathological morphology and inflammatory score, serum TgAb, TPOAb concentrations, and in vitro stimulated proliferation levels of splenic T lymphocytes. The preventive effect of immune tolerance induced by oral, subcutaneous or intramuscular injection of porcine thyroid freeze-dried powder on the onset of EAT in adult mice and the intervention effect on the imbalance of Th17 / Treg cells in EAT were observed.

[0068] The specific steps include:

[0069] Step 3.1: When mice are 8 weeks old, 100 μL PBS containing 1000 μg porcine thyroid freeze-dried powder is injected into the tail vein on days 0, 7, and 14. 100 μL PBS containing 100 μg LPS (lipopolysaccharide) is injected at intervals of 3 hours.

[0070] Step 3.2: The mice were killed at 12 weeks of age, and the tolerance effect was measured based on the thyroid pathological morphology and EAT (experimental autoimmune thyroiditis) score, serum TgAb, TPOAb concentrations, and in vitro stimulated proliferation level (SI) of splenic T lymphocytes.

[0071] Among them, the specific implementation of mouse thyroid tissue pathological examination and EAT model scoring is as follows:

[0072] (I) Mouse anatomical processing and specimen collection

[0073] (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.

[0074] (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.

[0075] (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.

[0076] (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:

[0077] Table 1 Scoring criteria for thyroid pathology in the mouse EAT model

[0078]

[0079] See also Figure 3 , Figure 3 The following are the HE staining images of mouse thyroid gland (200×, scale bar 100 μm) and the scatter plots of pathological evaluation of thyroid inflammation in each group of mice. Figure 3(a) is a staining image of the mouse EAT model with a thyroid pathology score of 0. Figure 3 (b) is a staining image of the mouse EAT model with a thyroid pathology score of grade 1. Figure 3 (c) is a staining image of the mouse EAT model with a thyroid pathology score of grade 2. Figure 3 (d) is a staining image of the mouse EAT model with a thyroid pathology score of 3. There was no lymph node infiltration in the control group, and the EAT score was 0, corresponding to Figure 3 (a) There was relatively little or no lymphocyte infiltration in the thyroid tissue of the PO and SC groups, and the EAT score was 0 or 1, corresponding to Figure 3 (a) and Figure 3 (b) Pathological results of mice in the pTLP group showed obvious lymphocyte infiltration in the thyroid gland, accompanied by destruction and atrophy of thyroid follicles. The EAT score was 2 or 3, corresponding to Figure 3 (c) and Figure 3 (d) If Figure 3 As shown in (e), the lymphocyte infiltration score of mice in the pTLP group was significantly higher than that of normal mice (Wilcoxon rank sum test P < 0.0001). Oral or subcutaneous injection of porcine thyroid freeze-dried powder within 0-24 hours after birth tended to inhibit the severity of thyroiditis, from a median of 2 in the pTLP group (igr from 1.75 to 2.75) to a median of 0.5 in the PO and SC groups (igr from 0 to 1) (Wilcoxon rank sum test P < 0.0001). Histopathology showed that the inflammation score of the IM group was between 1 and 2, which was lower than that of the pTLP group, but not statistically significant.

[0080] Furthermore, the specific operation of detecting the level of thyroid autoantibodies in mouse serum is as follows:

[0081] (A) ELISA method was used to detect the relative concentration of serum TgAb.

[0082] (1) Initial screening of positive serum, specifically including the following steps:

[0083] (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;

[0084] (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;

[0085] (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.

[0086] (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;

[0087] (e) Take out the ELISA plate, discard the serum samples in the wells, and wash three times;

[0088] (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;

[0089] (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;

[0090] (h) Immediately add 50 μL of 2Mol / L sulfuric acid to each well to stop color development;

[0091] (i) Immediately use a microplate reader to read the OD (optical density) value of each well at a wavelength of 450 nm;

[0092] (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 of the serum sample of the control mouse (mean ± 2 times the standard deviation). If the OD value of the modeling group exceeded the upper limit, it was defined as positive, so as to preliminarily screen out the mouse serum samples with positive TgAb.

[0093] (2) Drawing a TgAb concentration standard curve, specifically comprising the following steps:

[0094] (a) Take 100 μL of each of three mouse TgAb positive sera screened in the previous step and mix them evenly;

[0095] (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;

[0096] (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.

[0097] (II) ELISA method for detecting serum TPOAb, specifically including the following steps:

[0098] (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;

[0099] (2) Except for the blank well, 100 μL of HRP-labeled antibody was added to each well, sealed with a sealing film, and incubated in a 37°C incubator for 1 h;

[0100] (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;

[0101] (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;

[0102] (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.

[0103] See also Figure 4 , Figure 4 The relative concentration bar graphs of TgAb and TPOAb in the serum of each group of mice are shown in Figure 2. Figure 4 (a) is a bar graph of the relative concentration of serum TgAb in each group of mice; Figure 4 (b) is a bar graph of the relative concentration of TPOAb in the serum of each group of mice. Figure 4 As shown in (a), compared with the control group, all mice immunized with porcine thyroid freeze-dried powder or porcine thyroglobulin had increased thyroglobulin antibodies. Figure 4As shown in (b), the pTLP group also showed similar performance in thyroid peroxidase antibodies compared with the control group. In addition, oral and subcutaneous injection of porcine thyroid freeze-dried powder was more effective than intramuscular injection in preventing the development of thyroiditis. Compared with the pTLP group, the levels of TgAb and TPOAb in the PO, IM and SC groups were all reduced. Compared with the IM group, the decrease was more obvious in the PO and SC groups. The results suggest that neonatal administration of porcine thyroid freeze-dried powder can effectively prevent the occurrence of Hashimoto's thyroiditis. In addition, oral and subcutaneous administration is more effective than intramuscular administration in inducing immune tolerance. The possible mechanism is that there are fewer antigen-presenting cells in the muscle than in the skin and mucosa, and more antigens or multiple immunizations are required to achieve a more ideal protective effect.

[0104] Furthermore, the specific operations of in vitro culture and proliferation stimulation of mouse spleen T lymphocytes are as follows:

[0105] The following operations were all performed under a sterile environment.

[0106] (I) Preparation of spleen single cell suspension

[0107] (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.

[0108] (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;

[0109] (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.

[0110] (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;

[0111] (5) Add 10 mL of PBS solution to dilute and mix, and let stand at room temperature for 1 min;

[0112] (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;

[0113] (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.

[0114] (II) In vitro stimulation and proliferation of splenic T lymphocytes

[0115] (1) Take the above 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;

[0116] (2) repeating the above step (1), replacing the basal proliferation medium with T lymphocyte stimulation proliferation medium;

[0117] (3) placing the two 96-well cell culture plates in a 37° C., 5% CO 2 cell culture incubator and culturing for 72 h;

[0118] (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;

[0119] (5) Take out the 96-well plate and use an ELISA reader to measure the OD value of each well at 450 nm;

[0120] (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.

[0121] See also Figure 5 , Figure 5 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 5 a is the flow cytometry graph of the proportion of Th17 and Treg cells in the spleen of each group of mice; Figure 5 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.

[0122] To investigate whether these lymphocyte subsets play a role in pre-treatment mice leading to Tg and TPO antibodies and hypothyroidism, this example measured the proportions of these lymphocyte subsets in the spleens of related mice that died 4 weeks after the initial injection. Flow cytometry analysis showed that the proportion of TH17 in the pTLP group was higher than that in the control group (p<0.05, Figure 5 a). The proportion of TH17 in the OP and SC groups was lower than that in the pTLP group (p<0.05, Figure 5 b) The proportion of Treg cells showed completely opposite results, suggesting that the function of Treg cells is opposite to that of Th17 cells.

[0123] The present invention provides a drug for preventing Hashimoto's thyroiditis, and a preparation method and application thereof. The drug is a 10ug / ul sterile porcine thyroid lyophilized powder solution, and the active ingredients of the drug are thyroglobulin and thyroid peroxidase. Neonatal CBA / J mice can be induced to have immune tolerance by oral or subcutaneous injection of porcine thyroid lyophilized powder to prevent the occurrence of autoimmune thyroiditis in adulthood. Neonatal tolerance can prevent the occurrence of Hashimoto's thyroiditis. This immunoprevention method has high clinical practicality and operability, and provides a new direction for the immunoprevention of Hashimoto's thyroiditis. These data provide a strong explanation for the selection of antigen doses, antigen administration methods, and time windows in which hosts are prone to neonatal tolerance in future clinical work to prevent Hashimoto's thyroiditis through neonatal tolerance.

[0124] By detecting the proportion of Th17 (CD4+IL-17A+) and Treg (CD4+CD25+Foxp3+) cells in the spleen of mice, it was confirmed that oral administration and subcutaneous injection of porcine thyroid freeze-dried powder in the neonatal period to induce immune tolerance has a preventive effect on the onset of autoimmune thyroiditis in adult mice, which can increase the number of Treg cells in the spleen of mice, reduce the number of Th17 cells, and reshape the balance of Th17 / Treg cells. This discovery provides a relatively solid theoretical basis and experimental data as support for future in-depth research on the immune tolerance mechanism of Hashimoto's thyroiditis.

[0125] 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 preparing a drug for preventing Hashimoto's thyroiditis, characterized in that: include: S1: Thyroid tissue was isolated from the neck of freshly slaughtered pigs; S2: 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; S3: pre-treating the thyroid extract by freezing the thyroid extract overnight and placing the thyroid extract in liquid nitrogen for a predetermined time; S4: freeze-dry the pre-treated thyroid extract to obtain pig thyroid freeze-dried powder and store it in a frozen state.

2. The method for preparing the drug for preventing Hashimoto's thyroiditis according to claim 1, characterized in that: The S1 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.

3. The method for preparing the drug for preventing Hashimoto's thyroiditis according to claim 1, characterized in that: The S2 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.

4. The method for preparing the drug for preventing Hashimoto's thyroiditis according to claim 1, characterized in that: S3 includes: The thyroid extract was placed in a -80°C freezer overnight, and then placed in liquid nitrogen for at least 1 hour to pre-treat the thyroid extract.

5. The method for preparing the drug for preventing Hashimoto's thyroiditis according to claim 1, characterized in that: Also includes: 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. A drug for preventing Hashimoto's thyroiditis, characterized in that: The drug is prepared by the preparation method according to any one of claims 1 to 5, wherein the drug is a 10ug / uL sterile porcine thyroid freeze-dried powder solution, and the active ingredients of the drug are thyroglobulin and thyroid peroxidase.

7. The drug for preventing Hashimoto's thyroiditis according to claim 6, characterized in that The drug can be used within 24 hours of the neonatal period, and can be used orally, by subcutaneous injection, or by intramuscular injection.

8. Use of the drug for preventing Hashimoto's thyroiditis according to claims 1 to 5 for inducing tolerance in newborn mice.

9. The use according to claim 8, characterized in that: include: Select EAT susceptible strain CBA / J newborn mice, and orally, subcutaneously or intramuscularly inject the mice with a dose of 60ug / g body weight and a concentration of 10ug / uL of sterile porcine thyroid freeze-dried powder solution within 24 hours after birth.

10. The use according to claim 9, characterized in that: Also includes: On days 0, 7, and 14 after the mice reached 8 weeks of age, 100 μL of phosphate-buffered saline solution containing 1000 μg of porcine thyroid freeze-dried powder was injected into the tail vein, and 100 μL of phosphate-buffered saline solution containing 100 μg of lipopolysaccharide was injected at intervals of 3 h. Thyroid pathological morphology, EAT score, serum TgAb, TPOAb concentrations and in vitro stimulated proliferation levels of splenic T lymphocytes were obtained when the mice were 12 weeks old to measure the tolerance effect.

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