CN115606550A

CN115606550A - Construction method of animal model with low ovarian reserve function induced by autoimmune thyroiditis

Info

Publication number: CN115606550A
Application number: CN202211337630.9A
Authority: CN
Inventors: 陆华; 李方远; 张琦; 谭沛; 李小青; 陈仁; 刘芊辰; 杨茜; 强玲侠
Original assignee: Chengdu University of Traditional Chinese Medicine
Current assignee: Chengdu University of Traditional Chinese Medicine
Priority date: 2022-10-28
Filing date: 2022-10-28
Publication date: 2023-01-17
Anticipated expiration: 2042-10-28
Also published as: CN115606550B

Abstract

The invention relates to a method for constructing an animal model with low ovarian reserve function induced by autoimmune thyroiditis, which comprises the steps of taking an animal, injecting a primary immune emulsifier subcutaneously for 2 weeks continuously, 1-2 times per week, and injecting a reinforced immune emulsifier for 5-13 weeks from the 3 rd week and 1-2 times per week. The DOR model caused by EAT established by the invention has obvious thyroid pathological change and ovary reserve function reduction, and can be used for the research of autoimmune thyroiditis combined with low ovary reserve function diseases.

Description

Construction method of animal model with low ovarian reserve function induced by autoimmune thyroiditis

Technical Field

The invention relates to the technical field of biology, and in particular relates to a construction method of an animal model with low ovarian reserve function induced by autoimmune thyroiditis.

Background

The incidence of autoimmune thyroiditis is on the rising trend year by year due to factors such as environment and living pressure. Results from the study and Meta analysis suggest that women of child bearing age with autoimmune thyroiditis (AIT) have lower ovarian reserve function than women of child bearing age without autoimmune thyroiditis. In addition to the familial susceptibility, development of AIT has claimed that high iodine is the major causative factor, iodine being a strong oxidant that causes oxidative stress in the thyroid gland.

At present, animal models of autoimmune thyroiditis exist, but animal models of ovarian reserve hypofunction induced by autoimmune thyroiditis do not exist.

Disclosure of Invention

In order to solve the technical problem, the invention provides a method for constructing an animal model with low ovarian reserve function induced by autoimmune thyroiditis, which comprises the following steps:

taking animals, injecting a primary immune emulsifier subcutaneously for 2 weeks continuously, 1-2 times per week, injecting a reinforced immune emulsifier for 5-13 weeks from the 3 rd week, 1-2 times per week;

feeding said animal with sodium iodide solution daily during the injection of the emulsifier;

the primary immune emulsifier is prepared by mixing a pig thyroglobulin antigen solution with Freund's complete adjuvant, and the concentration of the pig thyroglobulin antigen is 200-800 mg.L ^-1 The emulsion of (4);

the intensified immune emulsifier is prepared by mixing a pig thyroglobulin antigen solution with Freund's incomplete adjuvant, and the concentration of the pig thyroglobulin antigen is 200-800 mg.L ^-1 The emulsion of (4).

Further, the time of injection was monday and thursday each week.

Further, the time of injection was started every monday and thursday at 9 am.

Further, the animal is a mammal, preferably a mouse.

Still further, the mouse is a Kunming mouse, preferably a Kunming mouse of 4-7 months of age.

Further, the site of injection is the neck, back, inner thighs and/or abdomen.

Further, the injection of the immune-enhancing emulsifier is performed for 5 weeks, 7 weeks, 9 weeks, 11 weeks or 13 weeks, preferably 11 weeks.

Further, the injection of the immune-enhancing emulsifier is carried out for 13 weeks, and then the feeding of the sodium iodide solution is continued until 17-19 weeks.

Further, the concentration of the sodium iodide solution is 0.3 to 1 g.L ^-1 。

Further, the preparation method of the primary immune emulsifier comprises the following steps:

freund's complete adjuvant and porcine thyroglobulin antigen solution were mixed at a ratio of 1:1 volume ratio, and mixing at 1000-5000 r/min to obtain viscous emulsifier with porcine thyroglobulin antigen concentration of 500 mg.L ^-1 。

Further, the preparation method of the booster immune emulsifier comprises the following steps:

freund's incomplete adjuvant and porcine thyroglobulin antigen solution were mixed at a ratio of 1:1 volume ratio, and mixing the mixture into a viscous emulsifier under the condition of the rotating speed of 1000-5000 r/min, wherein the concentration of the porcine thyroglobulin antigen is 500 mg.L ^-1 。

Furthermore, the pig thyroglobulin antigen solution is prepared by dissolving pig thyroglobulin antigen in phosphate buffer solution, and the concentration of the pig thyroglobulin antigen solution is 0.5-1 mg/ml.

The invention also provides an animal model with low ovarian reserve function induced by autoimmune thyroiditis, which is a mouse model constructed according to the method.

The invention establishes DOR with low ovarian reserve function caused by EAT of autoimmune thyroiditis with normal thyroid function by high iodine water feeding in combination with antigen immunity induction for 2 times a week for 11-13 weeks, and establishes DOR caused by EAT of hypothyroidism for more than 15 weeks. High iodine water feeding combined with 1 antigen immunization induction per week resulted in establishment of normovolemic EAT-induced DOR for 13-15 weeks and hypothyroidism-induced DOR for more than 17 weeks. Antigen immunity induction matched with high iodine water feeding is carried out 2 times per week, the modeling of the EAT merged DOR model with normal thyropathy is relatively early, the damage to the thyroid gland is obvious, and the ovarian reserve is obviously reduced. The DOR model caused by EAT established by the invention has obvious thyroid pathological change and ovarian reserve function reduction, and can be used for the research of autoimmune thyroiditis with ovarian reserve function low diseases.

It will be apparent that various other modifications, substitutions and alterations can be made in the present invention without departing from the basic technical concept of the invention as described above, according to the common technical knowledge and common practice in the field.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 anal temperature changes of mice in each group after model creation

FIG. 2 ovarian organ index for group J and group BIW

FIG. 3J group thyroid pathology scores

FIG. 4BIW group thyroid HE staining

FIG. 5 groups of mice thyroid HE staining

FIG. 6J groups of mice, ovarian follicles at all levels and corpus luteum

FIG. 7BIW mice ovarian all levels of follicles and corpus luteum

FIG. 8 group J and BIW mice ovarian HE staining

FIG. 9 group J mice Elisa serum thyroid antibody, alpha function and sex hormone levels

FIG. 10 levels of Elisa serum thyroid antibodies, thyrotoxication and sex hormones in BIW group mice

FIG. 11 group J mice serum oxidative stress marker

FIG. 12BIW group mice serum oxidative stress markers

Detailed Description

Example 1 construction of an autoimmune thyroiditis-induced ovarian Reserve hypofunction animal model

1) Preparation of the solution

High iodine content water: mixing 0.64g sodium iodide crystal with 1L purified water to obtain a mixture with a concentration of 0.64 g.L ^-1 High iodine water;

antigen solution: dissolving porcine thyroglobulin (pTg) antigen (1 mg/ml) in Phosphate Buffered Saline (PBS);

primary immune emulsifier: freund's complete adjuvant (CFA) and antigen solution were mixed at a ratio of 1:1 volume ratio is respectively absorbed into 1 50ml centrifuge tube, the centrifuge tube is placed on a vortex oscillator and is vibrated at high speed (about 2000 r/min) for 40 minutes until a viscous emulsifier is formed, and the final concentration of the emulsifier reaches 500 mg.L ^-1 Is ready to use when being prepared;

and (3) a boosting immune emulsifier: freund's incomplete adjuvant (IFA) and antigen solution were prepared at a volume ratio of 1 ^-1 A water-in-oil emulsion.

2) Molding machine

Primary immunization: injecting 0.2ml of primary immune emulsifier into subcutaneous multiple points (neck, back, inner thigh, abdomen) of mice for 2 weeks continuously and 2 times per week (beginning at 9 am every monday and thursday);

enhancing immunity: the mice were injected with 0.2ml of the fortified immune emulsifier at several subcutaneous points from week 3 for 11 weeks, and were fed with high iodine water 2 times per week (beginning at 9 am every monday, thursday).

Example 2 construction of an autoimmune thyroiditis-induced ovarian hypofunction animal model

1) Preparation of the solution

The same as in example 1.

2) Molding machine

Primary immunization: subcutaneous injection of 0.2ml of primary immune emulsifier at multiple points (neck, back, inner thigh, abdomen) for 2 weeks, 2 times per week (beginning at 9 am every monday, thursday);

and (3) strengthening immunity: the mice are injected with 0.2ml of the intensified emulsifying agent at multiple subcutaneous points from week 3, and the intensified immunity is continued for 5 weeks, 2 times per week (beginning at 9 am every monday, thursday) and fed with high iodine water during the molding period.

Example 3 construction of an autoimmune thyroiditis-induced ovarian hypofunction animal model

1) Preparation of the solution

Same as example 1

2) Molding die

enhancing immunity: the mice were injected with 0.2ml of the enhanced immune emulsifier at multiple subcutaneous points from week 3 for 13 weeks, and were fed with high iodine water 2 times per week (beginning at 9 am every monday, thursday).

Example 4 construction of an autoimmune thyroiditis-induced ovarian hypofunction animal model

1) Preparation of the solution

The same as in example 1.

2) Molding die

Primary immunization: a total of 0.2ml of primary immune emulsifier was injected subcutaneously into mice at multiple points (neck, back, inner thigh, abdomen) for 2 weeks, 1 time per week (beginning at 9 am every monday or thursday);

enhancing immunity: 0.2ml of the intensified immune emulsifier is injected into each mouse at multiple subcutaneous points from week 3, the intensified immunity is continued for 11 weeks, 1 time per week (beginning at 9 am every monday or thursday), and the mice are fed with high iodine water during molding.

Example 5 construction of an autoimmune thyroiditis-induced ovarian hypofunction animal model

1) Preparation of the solution

The same as in example 1.

2) Molding die

Primary immunization: subcutaneous injection of 0.2ml of primary immune emulsifier at multiple points (neck, back, inner thigh, abdomen, etc.) for 2 weeks, 1 time per week (beginning at 9 am every monday or thursday);

enhancing immunity: 0.2ml of the intensified immune emulsifier is injected into each mouse at multiple subcutaneous points from week 3, the intensified immunity is continued for 5 weeks, 1 time per week (beginning at 9 am every monday or thursday), and the mice are fed with high iodine water during molding.

Example 6 construction of an autoimmune thyroiditis-induced ovarian hypofunction animal model

1) Preparation of the solution

The same as in example 1.

2) Molding machine

Primary immunization: injecting 0.2ml of primary immune emulsifier into subcutaneous multiple points (neck, back, inner thigh, abdomen, etc.) of mice for 2 weeks, 1 time per week (beginning at 9 am every monday or thursday); and (3) strengthening immunity: 0.2ml of the intensified immune emulsifier is injected into each mouse at multiple subcutaneous points from week 3, the intensified immunity is continued for 13 weeks, 1 time per week (beginning at 9 am every monday or thursday), and the mice are fed with high iodine water during molding.

Example 7 construction of an autoimmune thyroiditis-induced hypoovarian Reserve animal model

1) Preparation of the solution

The same as in example 1.

2) Molding die

enhancing immunity: the mice were injected subcutaneously with multiple booster emulsifiers at 0.2ml points at 3 weeks, for 13 weeks of booster immunization, 2 times per week (beginning at 9 am every monday, thursday), and fed with high-iodine water starting on the day of injection of the primary booster emulsifier, up to 17 or 19 weeks.

The advantageous effects of the present invention are described below by way of test examples.

Test example 1

1 materials of the experiment

1.1 Experimental animals

Kunming mouse, female, 8-9 weeks old, 96 mice, weight 25-30g, provided by WUDDAO MIO (license number: SCXK (Chuan) 2020030), and feed provided by WUDDAO MIO. The feed is raised in cages in gynecology laboratories of Chengdu Chinese medicine university. The feeding environment is well ventilated, the temperature is kept at 20-24 ℃, the relative humidity is 40% -79%, and the day and night are illuminated.

1.2 Experimental drugs and reagents

TABLE 1 Main experimental drugs and reagents

2 method of experiment

2.1 preparation of reagents

2.1.1 high iodine Water

Mixing 0.64g sodium iodide crystal with 1L purified water to obtain a mixture with a concentration of 0.64 g.L ^-1 High iodine water;

2.1.2 antigen solutions

Porcine thyroglobulin (pTg) antigen (1 mg/ml) was dissolved in Phosphate Buffered Saline (PBS).

2.1.3 Primary Immunoamulsifiers Freund's complete adjuvant (CFA) and antigen solution were mixed at a ratio of 1:1 volume ratio of the mixture is respectively absorbed into 1 50ml centrifuge tube, the centrifuge tube is placed on a vortex oscillator and is vibrated at high speed (about 2000 rpm) for 40 minutes until a viscous emulsifier is formed, the final concentration of the emulsifier reaches 500 mg.L < -1 >, and the emulsifier is prepared for use at present.

2.1.4 boost immune emulsifier Freund's incomplete adjuvant (IFA) and antigen solution were prepared as a 500 mg.L-1 water-in-oil emulsion in a 1.

2.2 Experimental grouping and modeling drug delivery

2.2.1 Experimental groups

After 96 Kunming female mice are adaptively fed for 1 week, the mice are numbered according to the weight and the size of the mice, and are divided into a J control group, a BIW control group, a J model group and a BIW model group by adopting a random digital table method. The J model group and the BIW model group are divided into 7 week groups (J1 group and BIW1 group), 9 week groups (J2 group and BIW2 group), 11 week groups (J3 group and BIW3 group), 13 week groups (J4 group and BIW4 group), 15 week groups (J5 group and BIW5 group), 17 week groups (J6 group and BIW6 group) and 19 week groups (J7 group and BIW7 group) according to the material taking time window, and each 7 subgroups account for 16 groups, and each group is 6.

2.2.2 Molding administration

2.2.2.1 dosage and method of administration

(1) Model set

Model group J (antigen immunization induction 1 times/week): the prepared PTG antigen was mixed with an adjuvant according to 1:1, mixing, fully grinding to prepare emulsion antigen, and placing in a refrigerator at 4 ℃ for later use. Primary immunization: injecting 0.2ml of primary immune emulsifier into mice subcutaneously at multiple points (neck, back, inner thigh, abdomen, etc.) for 2 weeks, 1 time per week (beginning at 9 am every week); enhancing immunity: each mouse was injected subcutaneously with 0.2ml of booster emulsifier at multiple points starting at week 3 for 5-13 weeks, 1 time per week (starting at 9 am on a weekly basis). Antigen immune induction was stopped at 17 weeks and 19 weeks. During the molding period, the high iodine water is used for feeding.

BIW model group (2 antigen immunizations/week): the prepared PTG antigen was mixed with an adjuvant according to 1:1, mixing, fully grinding to prepare emulsion antigen, and placing the emulsion antigen in a refrigerator at 4 ℃ for later use. Primary immunization: injecting 0.2ml of primary immune emulsifier into subcutaneous multiple points (neck, back, inner thigh, abdomen, etc.) of mice for 2 weeks continuously and 2 times per week (beginning at 9 am every monday, thursday); enhancing immunity: 0.2ml of the booster emulsifier was injected subcutaneously at multiple points into each mouse from week 3, and the booster immunization was continued for 5-13 weeks 2 times a week (beginning at 9 am every monday, thursday). Antigen immunity induction was stopped at 17 weeks and 19 weeks. The high iodine water is used for feeding during the molding process.

(2) Control group

J control group: mice were injected subcutaneously (neck, back, inner thigh, abdomen, etc.) with 0.2ml of PBS buffer at multiple points, 1 time per week (9 am once a week).

BIW control group: mice were injected subcutaneously (neck, back, inner thigh, abdomen, etc.) with 0.2ml of PBS buffer at multiple points 2 times per week (9 am, once a week). During the molding period, general conditions of each group of mice such as body state, activity condition, hair color, anal temperature, weight and stool change are observed, and vaginal smears are collected every other day to observe the estrus cycle condition of each group of mice.

2.2.2.2 modeling and batch drawing

TABLE 3 modeling and batch sampling table

2.3 specimen Collection

2.3.1 preparation before specimen Collection all mice were observed by vaginal smear and sacrificed for material collection at the prophase of estrus.

2.3.2 blood sample collection after mouse anesthesia, cutting off two sides of mouse beard, collecting blood by orbital venous plexus blood sampling method, collecting blood sample in PCR tube, standing in refrigerator at 4 deg.C, centrifuging at 3000rpm for 10min, carefully absorbing upper mouse serum by pipettor, separating to obtain serum and plasma, transferring to labeled cryopreservation tube, and storing in ultralow temperature refrigerator at-80 deg.C.

2.3.3 tissue specimen Collection

The mice were sacrificed by decapitation, fixed on an ice plate in a supine position, the neck of the mice was sterilized, the trachea was separated and the thyroid gland was peeled off, the fascia and connective tissue were removed, and the thyroid tissue was fixed in 4% paraformaldehyde fixing solution for HE staining of the thyroid tissue. Cutting along the midline of the lower abdomen, searching the Y-shaped uterus of a rat along the vaginal orifice, searching the left ovary and the right ovary upwards along two angles of the uterus, carefully peeling off the fat tissues around the uterus and the ovary, weighing the wet weight of the uterus and the ovary, respectively putting the uterus and the left ovary into an EP tube, and quickly freezing by liquid nitrogen to be detected. One side of the ovaries was fixed in 4% paraformaldehyde fixing solution for HE staining.

2.4 Observation index

2.4.1 general conditions

The mice in each group were observed daily for survival, mental status, behavioral activities, eating, hair cover (with or without standing hair, fluffy body hair, depilation), excretion of feces, etc. Body weight and anal temperature were recorded every friday. The general state of the mice was observed during the experiment and changes were recorded. If any animal died, the number and cause of the death was recorded.

2.4.2 estrus cycle

2.4.2.1 estrus cycle observations

Mice were smeared with vaginal shed cells every morning from 8 to 9, as follows: wetting a sharp-pointed cotton swab special for tattooing with 0.9% NaCl solution, inserting into mouse vagina for about 0.5cm, rotating clockwise for 3-4 weeks, extracting the cotton swab, rotationally coating vaginal shedding on the cotton swab on a slide (without overlapping), placing the slide under a COIC microscope (10 x10 times) and observing cell morphology, and judging the change of the estrus cycle of the mouse. The estrus cycle of the sexually mature female mice is 4-5 days, and is divided into prophase estrus, anaphase estrus and estrus interval according to the morphological change of cytological smears. A cycle that is too long (. Gtoreq.6 d), incomplete, too short, or long in the same cycle (. Gtoreq.3 d) is considered to be a cycle disorder.

2.4.2.2 vaginal shed cell characteristics

TABLE 4 characteristics of vaginal shed cells

2.4.3 histological observations of thyroid and ovary

2.4.3.1 thyroid and ovarian HE staining method

(1) Embedding: dehydrating the fixed tissue by a full-automatic dehydrator (the dehydration time is 4h of 75% alcohol, 2h of 85% alcohol, 1h of 95% alcohol, 0.5h of 100% alcohol, 0.5h of 100% alcohol, 10min of dimethylbenzene, 1h of paraffin, 2h of paraffin and 3h of paraffin), and embedding;

(2) Slicing: cutting the tissue into thin slices with the thickness of 5 mu m by adopting a LeicaRM2235 microtome, flattening the tissue in warm water, fishing the tissue on a glass slide, and baking and slicing the tissue for at least 2 hours at the temperature of 60 ℃;

(3) Dyeing: staining with hematoxylin for 10-20min; washing with tap water for 1-3min; differentiating with hydrochloric acid alcohol for 5-10s; washing with tap water for 1-3min; putting into warm water of 50 deg.C or weakly alkaline aqueous solution to turn blue until blue color appears; washing with tap water for 1-3min; adding 85% alcohol for 3-5min; staining with eosin for 3-5min; washing with water for 3-5s; gradient alcohol dehydration; the xylene is transparent; sealing with neutral gum;

(4) Microscopic examination: mouse ovarian tissue image acquisition of the sections was performed using a Pannoramic 250 digital section scanner produced by 3DHISTECH (Hungary). The mouse thyroid tissue adopts a BA210Digital three-mesh shooting microscopic imaging system produced by Miaodi industry group, inc. to collect images of the slices. Each section is used for observing the whole tissue before 40 times, general lesions are observed, and 100 times and 400 times of images are selected from the region to be observed to observe specific lesions.

Thyroid pathology score thyroid histopathology score reference Charveire classification method

TABLE 5Charveire classification

2.4.3.3 stage follicle count

And counting the number of follicles at each stage, namely primordial follicles, primary follicles, secondary follicles, mature follicles, atretic follicles, a corpus luteum and the like.

2.4.4 determination of serum thyroid antibody, thyroidism, AMH and oxidative stress marker by enzyme-linked immunosorbent assay (ELISA)

2.4.4.1 rewarming all reagents to room temperature.

2.4.4.2 sample adding of the standard substance, a standard substance hole and a sample hole are arranged, and 50 mu L of standard substance with different concentrations is added into each standard substance hole.

2.4.4.3 blank wells (blank reference wells containing no sample and enzyme-labeled reagent, and the rest wells operating in the same manner) and sample wells to be detected are provided for sample addition. And (3) adding 40 mu L of sample diluent into the sample hole to be detected on the enzyme-labeled coated plate, and then adding 10 mu L of sample to be detected (the final dilution of the sample is 5 times).

2.4.4.4 incubation with enzyme

Add enzyme labeling reagent 100. Mu.L to each well except blank wells, seal plate with sealing plate, and incubate at 37 ℃ for 60 minutes.

2.4.4.5 preparing the solution, diluting 20 times of the concentrated washing solution with 20 times of distilled water for later use.

2.4.4.6 washing, carefully uncovering the sealing plate film, discarding the liquid, spin-drying, filling washing liquid in each hole, standing for 1min, discarding, repeating the steps for 5 times, and patting dry.

2.4.4.7 developing, adding 50 μ L of substrate solution A into each well, adding 50 μ L of substrate solution B, shaking gently, mixing, and developing at 37 deg.C in dark for 15min.

2.4.4.8 stop the addition of 50. Mu.1 stop solution per well, at which point the blue color turned immediately yellow.

2.4.4.9 determination the absorbance (OD value) of each well was measured sequentially at a wavelength of 450nm within 15min.

2.5 statistical analysis

ˉ

Measuring data: expressed as mean and standard deviation (X ± SD), ANOVA single-dose data were used if the data fit the normal distribution, uniform variance: the mean and standard deviation (LSD test in the factor variance analysis is used for multiple comparison among groups, if the variance is not uniform, the Games-Howelltest is used for multiple comparison among groups, the California test is used for counting data, and all statistical tests adopt bilateral test, when P is less than 0.01, the difference has statistical significance, and when P is less than 0.05, the difference has statistical significance, and when P is more than 0.05, the difference has no statistical significance.

3 results of the experiment

During the molding process, some mice die, and the causes of death may be as follows: induration and suppuration infection in the slow release process of the injection part medicament, occult subcutaneous bleeding caused by needle prick and blood vessels of the injection, and the like.

TABLE 6 number of samples (n) before and after modeling of each group of mice

3.1 general conditions in groups of mice

Mice in the BIW control group and the J control group have normal activities, hair luster, normal defecation and regular estrus cycle. After the model group mice are molded and injected subcutaneously at multiple points, the injection part is hardened and still does not fade after the molding is finished; the stool color is light and thin beginning at week 4; approximately 60% of mice had neck, perilabial, back, etc. hair loss beginning at approximately week 7 of molding; intestinal dysfunction, and laxity and defecation when the intestinal dysfunction is stimulated to the perianal area in the vaginal smear process.

3.1.1 weight and anal temperature changes in groups of mice

Table 7.J group mice body weight statistics

Note: model group J compared to control group J: * P <0.05,. P <0.01; comparison between model groups: because the number of model groups is large, in order to avoid confusion caused by excessive symbols, differences are respectively represented by 'delta' plus the serial number of the group, namely: comparison with group J1: Δ 1P < -0.05 and Δ 1P < -0.01; comparison with group J2: Δ 2P < -0.05 and Δ Δ 2P < -0.01; comparison with group J3: Δ 3P-cloth 0.05, and Δ 3P-cloth 0.01; comparison with group J4: 0.05 of delta 4P (P) -knot, 0.01 of delta 4P-knot; comparison with group J5: Δ 5p yarn-woven fabric 0.05, Δ 5p yarn-woven fabric 0.01; comparison with group J6: Δ 6P-s-0.05, Δ Δ 6P-s-0.01; comparison with group J7: Δ 7p tres 0.05, Δ 7p tres 0.01.

The weights and anal temperatures of the mice in the group J are in accordance with normal distribution and the variance is homogeneous, and the LSD test in one-way ANOVA is adopted.

(1) Weight: differences between body weights of J mice before modeling were not statistically significant (P > 0.05). Compared with the control group, the weight of the model group mice after model building is obviously reduced in the J1 group and the J2 group, and the difference has statistical significance (P is less than 0.05); group J3-7 had significantly reduced body weight, with statistically significant differences (P < 0.01); no statistical difference in body weight was apparent between the J model groups (J1-J7) (P > 0.05).

(2) Anal temperature: differences between anal temperatures in J mice before molding were not statistically significant (P > 0.05). Compared with a control group, the model group has a reduced trend in J1, J2 and J3, but the difference is not statistically different (P is more than 0.05); the anal temperature of the J4 group and the J5 group is obviously reduced, and the difference is statistical (P is less than 0.05); the anal temperature between the J6 group and the J7 group is obviously reduced, and the difference is statistically different (P < 0.01).

TABLE 8 statistical results of body weights of BIW group mice

Note: BIW model group compared with BIW control group<0.05，**P<0.01; comparison between model groups, compared to BIW1 group: ^Δ1 P<0.05； ^ΔΔ1 P<0.01; comparison with BIW2 group: ^Δ2 P<0.05， ^ΔΔ2 P<0.01; comparison with BIW3 group: ^Δ3 P<0.05； ^ΔΔ3 P<0.01; (ii) a Comparison with BIW4 group: ^Δ4 P<0.05， ^ΔΔ4 P<0.01; comparison with BIW5 group: ^Δ5 P<0.05， ^ΔΔ5 P<0.01; comparison with BIW6 group: ^Δ6 P<0.05， ^ΔΔ6 P<0.05; comparison with BIW7 group: ^Δ7 P<0.05， ^ΔΔ7 P<0.01。

the weight and anal temperature of the BIW group mice are in accordance with normal distribution and the variance is homogeneous, and the LSD test in one-way ANOVA is adopted.

(1) Weight: no significant difference (P > 0.05) exists among the body weight groups of BIW mice before model building. Compared with the control group, the BIW1 group has statistical difference in obvious weight loss (P < 0.05) of the model group mice after model building; (ii) a The weight loss of BIW2-BIW7 groups is very obvious in difference, and the difference is statistical (P is less than 0.01); no statistical difference in body weight was observed between BIW1-BIW7 groups in the model group (P > 0.05).

(2) Anal temperature: no significant difference (P is more than 0.05) exists among anus temperature groups of BIW groups before model building. After modeling, the anal temperature of the mice in the model group is compared with that of the control group, and the BIW1 group has no statistical difference (P is more than 0.05); the anal temperature of BIW2 group, BIW3 group and BIW4 group is obviously reduced, and the difference has statistical difference (P is less than 0.05); the anal temperature between BIW5, BIW6 and BIW7 groups is obviously reduced, and the difference is statistically different (P < 0.01). The anal temperature of the J and BIW mice gradually decreased with the time of molding, as shown in FIG. 1.

3.1.2 groups of mice have periodic changes in estrus

TABLE 9 estrous periodic variation during modeling of J groups of mice

Note: the cycle of the estrus of the mice is 4-6d, and the cycle is overlong (more than or equal to 6 d), incomplete, too short or long in the same cycle (more than or equal to 3 d) is considered as cycle disorder. Delta estrus prophase; o represents estrus; the army is in the anaphase; o estrus phase.

Group J vaginal smears showed: j the estrous cycle of the control group has no obvious change; the J model group has no obvious disorder in the estrus period within 1-2 weeks of molding; in 3-6 weeks of model making, the disturbance rate of the estrus cycle of the model group is 16.67% -33.33%; the disturbance rate of the estrus cycle of the model group is 33.33-83.33% in 7-15 weeks of molding; the periodic disorder rate of the group is between 80.00 and 83.33 percent after the high iodine water is continuously fed for 16 to 19 weeks.

TABLE 10 estrous cycle changes during modeling of BIW group mice

Note: the cycle of the estrus of the mice is 4-6d, and the cycle is overlong (more than or equal to 6 d), incomplete, too short or long in the same cycle (more than or equal to 3 d) is considered as cycle disorder. Delta estrus prophase; o represents estrus; the estrus period; estrus interval.

BIW group vaginal smear show: the estrus cycle of the BIW control group has no obvious change; the BIW model group has no obvious disorder in the estrus period within 1-2 weeks of molding; in 3-6 weeks of molding, the disturbance rate of the estrus cycle of the BIW model group is 16.70% -50.00%; the disturbance rate of the estrus cycle of the BIW model group is 50.00-80.00% in 7-15 weeks of molding; in 16-19 weeks of the molding by continuously feeding the BIW model group with high iodine water, the estrus cycle disorder rate is 80.00% -83.33%.

3.1.3 gonadal indices in groups of mice

The mouse ovarian index is calculated according to the following formula: ovarian index = wet weight of bilateral ovaries (mg)/weight of mice before sacrifice (g) x100%.

TABLE 11 ovarian organ index profiles in groups of mice

Note: the J model was compared to the J control group, ^* P<0.05， ^** P<0.01; model group comparisons, to J1 group: ^Δ1 P<0.05， ^ΔΔ1 P<0.01; comparison with group J2: ^Δ2 P<0.05， ^ΔΔ2 P<0.01; comparison with group J3: ^Δ3 P<0.05， ^ΔΔ3 P<0.01; comparison with group J4: ^Δ4 P<0.05， ^ΔΔ4 P<0.01; comparison with group J5: ^Δ5 P<0.05， ^ΔΔ5 P<0.01; comparison with group J6: ^Δ6 P ^<0.05 ， ^ΔΔ6 P<0.01; comparison with group J7: ^Δ7 P<0.05， ^ΔΔ7 P<0.01. the BIW model group was compared with the BIW control group, ^* P<0.05， ^** P<0.01; model group comparisons, to BIW1 group: ^Δ1 P<0.05， ^ΔΔ1 P<0.01; comparison with BIW2 group: ^Δ2 P<0.05， ^ΔΔ2 P<0.01; comparison with BIW3 group: ^Δ3 P<0.05， ^ΔΔ3 P<0.01; comparison with BIW4 group: ^Δ4 P<0.05， ^ΔΔ4 P<0.01; comparison with BIW5 group: ^Δ ⁵ P<0.05， ^ΔΔ5 P<0.01; comparison with BIW6 group: ^Δ6 P<0.05， ^ΔΔ6 P<0.01; comparison with BIW7 group: ^Δ7 P<0.05， ^ΔΔ7 P<0.01。

the ovarian organ index of the J group mice conforms to normal distribution and the variance is homogeneous, and the LSD test in one-way ANOVA is adopted.

(1) Group J mice ovarian organ index: the ovarian index was reduced in the J1 group compared to the control group, but the difference was not statistically significant (P > 0.05); the J2 and J3 groups were significantly reduced (P < 0.05). The J4, J5, J6 and J7 groups were significantly reduced (P < 0.01). Comparison between model J groups: differences among groups J1, J2, J3 and J4 have no statistical significance (P > 0.05); the J5, J6 and J7 groups were all significantly lower (P < 0.01) than the J1, J2, J3 and J4 groups.

(2) Comparison among BIW group ovarian organ index groups: the ovary index of the BIW1 group is reduced compared with that of the control group, but the difference is not statistically significant (P is more than 0.05); the BIW2 group and the BIW3 group are obviously reduced (P is less than 0.05); BIW4, BIW5, BIW6, BIW7 groups were significantly reduced (P < 0.01). Comparison between BIW model groups: the differences among the BIW1 group, the BIW2 group, the BIW3 group and the BIW4 group have no statistical significance (P is more than 0.05); the BIW5 group is obviously reduced (P < 0.05) compared with the BIW4 group, and is obviously reduced (P < 0.01) compared with the BIW1 group, the BIW2 group and the BIW3 group; the BIW6 group and the BIW7 group are obviously reduced compared with the BIW1 group, the BIW2 group, the BIW3 group and the BIW4 group (P is less than 0.01). The ovarian organ index of the J group and BIW mice gradually decreased with the extension of the molding time, and the figure is shown in figure 2.

3.2 histological observations of thyroid and ovary in groups of mice

3.2.1 thyroid HE staining in groups of mice

Thyroid HE staining in 3.2.1.1J group

TABLE 12 statistical results for group J thyroid Charveire scores

Note: model groups (groups J1-7) were compared to the J control group, ^* P<0.05， ^** P<0.01; model group comparisons, to J1 group: ^Δ1 P<0.05， ^ΔΔ1 P<0.01; comparison with group J2: ^Δ2 P<0.05， ^ΔΔ2 P<0.01; comparison with group J3: ^Δ3 P<0.05， ^ΔΔ3 P<0.01; comparison with group J4: ^Δ4 P<0.05， ^ΔΔ4 P<0.01; comparison with group J5: ^Δ5 P<0.05， ^ΔΔ5 P<0.01; comparison with group J6: ^Δ6 P<0.05， ^ΔΔ6 P<0.01; comparison with group J7: ^Δ7 P<0.05， ^ΔΔ7 P<0.01。

the infiltration intensity of thyroid lymphocytes, thyroid follicular structure change and pathological gross variation of mice in the J group are different, and the statistical results are as follows by adopting Games-Howelltest test:

(1) Thyroid lymphocyte infiltration strength: compared with the J control group, no obvious pathological change is seen in the J1 group and the J2 group, and the thyroid lymphocyte infiltration intensity of the J3 group is increased (mice No. (1) (3) (6)), but the difference is not statistically different (P is more than 0.05); the J4 group, the J5 group, the J6 group and the J7 group are obviously higher (P is less than 0.01). Comparison between model J groups: the J4 group and the J5 group are obviously increased (P is less than 0.05) compared with the J1 group and the J2 group; group J6, group J7 significantly increased (P < 0.01) compared to group J1, group J2 and significantly increased (P < 0.05) compared to group J3; (ii) a The differences between the remaining groups were not statistically significant.

(2) Thyroid follicular structural changes: the thyroid follicular cell infiltration intensity was increased in group J3 compared to group J control by 60% in the (mice (No. 1) (3) (6)) molding rate, but the difference was not statistically different (P > 0.05); the J4 group, the J5 group, the J6 group and the J7 group are obviously increased (P < 0.01). Comparison between model J groups: the J4, J5, J6, J7 groups were significantly higher (P < 0.01) than the J1, J2, J3 groups, with no statistical difference between the remaining groups.

(3) Thyroid pathology gross score change, i.e. the thyroid lymphocyte infiltration intensity of the J3 group is increased compared with that of the J control group, but the difference is not statistically different (P is more than 0.05); the J4 group, the J5 group, the J6 group and the J7 group are obviously increased (P < 0.01). J model group comparisons: the group J4, J5, J6, J7 were significantly higher (P < 0.01) than the group J1, J2, J3, with no statistical significance for the differences between the remaining groups. The thyroid pathology scores in group J tended to increase gradually with the time of molding, as shown in fig. 3.

3.2.1.2BIW group thyroid HE staining

TABLE 13 statistical results of Charveire class score for thyroid gland in BIW group

Note: the model groups (BIW 1-7 groups) were compared with the BIW control group, ^* P<0.05， ^** P<0.01; model group comparisons, to BIW1 group: ^Δ1 P<0.05， ^ΔΔ1 P<0.01; comparison with BIW2 group: ^Δ2 P<0.05， ^ΔΔ2 P<0.01; comparison with BIW3 group: ^Δ3 P<0.05， ^ΔΔ3 P<0.01; comparison with BIW4 group: ^Δ4 P<0.05， ^ΔΔ4 P<0.01; comparison with BIW5 group: ^Δ5 P<0.05， ^ΔΔ5 P<0.01; comparison with BIW6 group： ^Δ6 P<0.05， ^ΔΔ6 P<0.01; comparison with BIW7 group: ^Δ7 P<0.05， ^ΔΔ7 P<0.01。

the infiltration intensity of thyroid lymphocytes, thyroid follicular structure change and pathological gross variation of mice in the BIW group are different, and the statistical results are as follows by adopting Games-Howelltest test:

(1) Thyroid lymphocyte infiltration strength: compared with the BIW control group, the infiltration intensity of the thyroid lymphocytes in the BIW1 group is not changed, the infiltration intensity of the thyroid lymphocytes in the BIW2 group is increased, and the mice (No. 4, no. 5 and No. 6) have lymphocytes and neutrophils, but the difference is not statistically different (P is more than 0.05); BIW3, BIW4, BIW5 groups were significantly elevated (P < 0.05); (ii) a BIW6 and BIW7 groups were significantly elevated (P < 0.01). Comparison between BIW model groups: BIW3, BIW4 and BIW5 were all significantly higher (P < 0.01) than BIW1, and the differences among the other groups were not statistically significant.

(2) Thyroid follicular structure change: compared with a BIW control group, the thyroid follicular structure of the BIW1 group (6) mice is slightly changed, the follicular structure of the thyroid glands of the BIW2 group (4), (5) and (6) mice is changed, and the molding rate is 50%; the scores of the BIW1 group and the BIW2 group are increased, but the difference is not statistically different (P is more than 0.05); BIW3 group was significantly higher (P < 0.05), BIW4 group, BIW5 group, BIW6 group, BIW7 group was significantly higher (P < 0.01). Comparison between BIW model groups: BIW6 and BIW7 groups were significantly higher than BIW1 and BIW2 groups; the differences between the remaining groups were not statistically significant (P > 0.05).

(3) Thyroid pathology gross score changes: the scores of the BIW1 group and the BIW2 group are increased compared with the BIW control group, but the difference is not statistically different (P is more than 0.05); BIW3, BIW4, BIW5, BIW6 and BIW7 groups were significantly higher (P < 0.01). Comparison between BIW groups: the BIW3 group, the BIW4 group and the BIW5 group are obviously increased (P is less than 0.05) compared with the BIW1 group, the BIW6 group and the BIW7 group are obviously increased (P is less than 0.01) compared with the BIW1 group and the BIW2 group, and the difference among the other groups has no statistical significance (P is more than 0.05). The thyroid pathology score of BIW model group has a gradually rising trend along with the extension of the molding time, and is shown in figure 4.

3.2.1.3 Graves HE staining histological changes in groups of mice

The thyroid cells of the control mice under the light lens are orderly arranged and distributed, the follicular is oval, more epithelial cells are cubic, the staining is light red, the cytoplasm is rich, the nucleus is round, the size is uniform, the follicular cavity contains rich colloid, and the control mice are free from lymphocyte infiltration and follicular damage. The thyroid follicular epithelium of the molded mouse with uneven thyroid gland size is low cubic, and has different colloid thicknesses, so that the thyroid follicular destruction and lymphocyte infiltration can be seen. See FIG. 5 for thyroid HE staining for each group

3.2.2 groups of mice ovarian HE staining

3.2.2.1J group mice ovarian HE staining

TABLE 14 number of ovarian follicles and corpus luteum in group J mice at all levels

Note: the J model group was compared with the J control group, ^* P<0.05， ^** P<0.01; comparison between model groups, and J1 group: ^Δ1 P<0.05， ^ΔΔ1 P<0.01; comparison with group J2: ^Δ2 P<0.05， ^ΔΔ2 P<0.01; comparison with group J3: ^Δ3 P<0.05， ^ΔΔ3 P<0.01; comparison with group J4: ^Δ4 P<0.05， ^ΔΔ4 P<0.01; comparison with group J5: ^Δ5 P<0.05， ^ΔΔ5 P<0.01; comparison with group J6: ^Δ6 P<0.05， ^ΔΔ6 P<0.01; comparison with group J7: ^Δ7 P<0.05， ^ΔΔ7 P<0.01。

except for primary follicles, all levels of follicle counts and corpus luteum of each group of mice are in accordance with normal distribution, all the variances of each group are uniform, so that LSD test in one-factor variance is adopted, and the primary follicles are tested by Games-Howelltest.

(1) Primordial follicles: compared with the J control group, primordial follicles of the J1 group, the J2 group and the J3 group are slightly reduced, but the difference is not statistically significant (P is more than 0.05); the J4 group and the J5 group are obviously reduced (P < 0.05); the primordial follicles in the J6 and J7 groups were significantly reduced (P < 0.01). J model group comparisons: primordial follicles were significantly reduced in group J7 compared to group J1 and group J2 (P < 0.05), and the differences among the remaining groups were not statistically different (P > 0.05).

(2) Primary follicle: compared with the J control group, the primary follicles in the J1 group, the J2 group and the J3 group were slightly decreased, but the differences were not statistically different (P > 0.05), and the primary follicles in the J4 group, the J5 group, the J6 group and the J7 group were significantly decreased (P < 0.05). J model group comparisons: the differences between groups were not statistically different (P > 0.05).

(3) Secondary follicles: compared with the J control group, the secondary follicles of the J1 group, the J2 group and the J3 group are slightly reduced, but the difference is not statistically different (P > 0.05), and the secondary follicles of the J4 group, the J5 group, the J6 group and the J7 group are obviously reduced (P < 0.05). J model group comparisons: the differences between groups were not statistically different (P > 0.05).

(4) Mature follicles: comparison between groups: the difference was not statistically significant (P > 0.05).

(5) Atretic follicle: compared with the J control group, the J1 group, the J2 group and the J3 group have increasing tendency, and the difference has no statistical significance (P is more than 0.05); the J4 group is obviously increased (P < 0.05), and the J5 group, the J6 group and the J7 group are obviously increased (P < 0.01); comparison between model groups: the J5 group and the J6 group are obviously increased compared with the J1 group (P is less than 0.05), the J5 group and the J6 group are obviously increased compared with the J2 group (P is less than 0.01), the J7 group is obviously increased compared with the J2 group (P is less than 0.05), and the difference among the other groups has no statistical significance (P is more than 0.05).

(6) Corpus luteum: compared with the J control group, the J1 group and the J2 group have a reduction trend, and the difference has no statistical significance (P is more than 0.05); the J3 group, the J4 group and the J5 group are obviously reduced (P is less than 0.05); the J6 group and the J7 group were significantly reduced (P < 0.01). Compared among J model groups, the difference is not statistically significant (P is more than 0.05). The number of follicles at each level and the number of corpus luteum in group J are shown in FIG. 6.

3.2.2.2BIW mice ovary HE staining

TABLE 15 ovarian follicle levels and corpus luteum numbers in BIW group mice

Note: the BIW model group was compared with the BIW control group, ^* P<0.05， ^** P<0.01; comparison between model groups, compared to BIW1 group: ^Δ1 P<0.05， ^ΔΔ1 P<0.01; comparison with BIW2 group: ^Δ2 P<0.05， ^ΔΔ2 P<0.01; comparison with BIW3 group: ^Δ3 P<0.05， ^ΔΔ ³ P<0.01; comparison with BIW4 group: ^Δ4 P<0.05， ^ΔΔ4 P<0.01; comparison with BIW5 group: ^Δ5 P<0.05， ^ΔΔ5 P<0.01; comparison with BIW6 group: ^Δ6 P<0.05， ^ΔΔ6 P<0.01; comparison with BIW7 group: ^Δ7 P<0.05， ^ΔΔ7 P<0.01。

all the follicles and corpus luteum in BIW group are in accordance with normal distribution and homogeneous variance, and LSD test in single-factor variance is adopted.

(1) Primordial follicles: compared with the BIW control group, the primordial follicles of the BIW1 group and the BIW2 group have a slightly reduced trend, but the difference is not statistically different (P is more than 0.05); BIW3 group was significantly reduced compared to it (P < 0.05); primordial follicles were significantly reduced in BIW4, BIW5, BIW6, BIW7 groups (P < 0.01). Compared among BIW model groups (BIW 1-7 groups), the difference is not statistically significant (P is more than 0.05).

(2) Primary follicles: compared with the BIW control group, the BIW1 group, the BIW2 group and the BIW3 group have slightly reduced tendency, but the difference is not statistically different (P is more than 0.05); the BIW4 group and the BIW5 group are obviously reduced (P is less than 0.05); BIW6 and BIW7 groups were significantly reduced (P < 0.01). Compared among BIW model groups (BIW 1-7 groups), the difference is not statistically significant (P is more than 0.05).

(3) Secondary follicles: compared with the BIW control group, the BIW1 group, the BIW2 group, the BIW3 group and the BIW4 group have a reduction trend, but the difference does not have statistical difference (P is more than 0.05); the BIW5, BIW6 and BIW7 groups were significantly reduced (P < 0.05). . The differences were not statistically significant (P > 0.05) when compared between BIW1-7 groups.

(4) Mature follicles: compared among groups, the difference is not statistically significant (P > 0.05).

(5) Atretic follicle: compared with the BIW control group, the BIW1 group and the BIW2 group have increasing tendency, and the difference has no statistical significance (P is more than 0.05); the BIW3 group, the BIW4 group and the BIW5 group are obviously increased (P is less than 0.05); the BIW6 group and the BIW7 group are remarkably increased (P is less than 0.01); compared among model groups, the difference is not statistically significant (P is more than 0.05).

(6) Corpus luteum: compared with the BIW control group, the BIW1 group has a reduction trend, and the difference has no statistical significance (P is more than 0.05); the BIW2 group and the BIW3 group are obviously reduced (P is less than 0.05); BIW4, BIW5, BIW6 and BIW7 groups were significantly reduced (P < 0.01). Compared with BIW1, BIW5, BIW6 and BIW7 groups were significantly reduced (P < 0.05), and the comparison difference among the rest groups was not statistically significant (P > 0.05). Ovarian follicular counts at all levels and the number of corpus luteum in mice in the BIW group are shown in FIG. 7. Ovarian HE staining in J and BIW mice is shown in figure 8.

3.3 determination of serum thyroid antibodies, thyroid function and reproductive hormone levels by enzyme-linked immunosorbent assay (ELISA)

Serum ELisa results of 3.3.1J group mice

TABLE 16 serum ELisa results of group J mice

the ELisa results of the group J sera are in accordance with normal distribution and homogeneity of variance, so the LSD test in one-wayANOVA is adopted.

(1) TGAb: compared with the J control group, the TGAb concentrations of the J1 group, the J2 group and the J3 group are increased but the difference is not statistically significant (P is more than 0.05); the concentration of J4 group and J6 group is obviously increased (P < 0.05); the concentration of J5 group and J7 group is obviously increased (P < 0.01). . Comparison between model J groups: the concentrations of the J5 group and the J7 group are obviously higher than that of the J2 group (P is less than 0.01), the concentration of the J4 group is obviously higher than that of the J2 group (P is less than 0.05), and the differences among the other groups have no statistical significance (P is more than 0.05).

(2) TPOAb: the concentrations of TPOAb in the J1, J2 and J3 groups were increased but the differences were not statistically significant (P > 0.05) compared to the J control group; the concentrations of the J4 group, the J5 group, the J6 group and the J7 group are obviously increased (P < 0.01); compared with the J model group, the concentrations of the J4 group, the J5 group, the J6 group and the J7 group are obviously increased compared with those of the J1 group and the J2 group (P is less than 0.01); the J4 group and the J7 group are obviously increased (P < 0.05) compared with the J3 group, and the J5 group is obviously increased (P < 0.01) compared with the J3 group; the differences between the remaining groups were not statistically significant (P > 0.05).

(3) FT3, compared with the J control group, FT3 levels of the J1 group, the J2 group, the J3 group, the J4 group and the J5 group are reduced, the difference is not significant, and the statistical significance is not achieved (P is more than 0.05); FT3 levels were significantly decreased in J6 and J7 groups (P < 0.05). Compared among the J model groups, the difference has no statistical significance (P > 0.05).

(4) FT4: compared with the J control group, the FT3 levels of the J1 group, the J2 group, the J3 group, the J4 group and the J5 group are reduced, the difference is not significant, and the statistical significance is not achieved (P is more than 0.05); FT4 levels of J6 group and J7 group are reduced, the difference is obvious (P < 0.05), compared with J model group, J6 group and J7 group are obviously reduced (P < 0.05) compared with J1 group, and the difference among the other groups has no statistical significance (P > 0.05).

(5) TSH: compared with a control group, TSH levels of the J1 group, the J2 group, the J3 group, the J4 group and the J5 group are increased, the difference is not significant, and the statistical significance is not achieved (P is more than 0.05); TSH levels were significantly elevated in J6 and J7 groups (P < 0.05). J model group comparisons: the J6 group was significantly higher (P < 0.05) than the J1 group, the J7 group was significantly higher (P < 0.01) than the J1 and J2 groups, and the differences among the other groups were not statistically significant (P > 0.05).

(6) AMH: compared with a control group, the AMH levels of the J1 group, the J2 group and the J3 group are reduced, and the difference has no statistical significance (P is more than 0.05); AMH levels were significantly decreased in groups J4, J5, J6, and J7 (P < 0.01). Compared with the J model group, the J5 group and the J7 group are obviously reduced compared with the J1 group (P is less than 0.05), and the difference among the other groups has no statistical significance (P is more than 0.05). The thyroid antibodies and TSH of the mice in the group J gradually increase along with the extension of the molding time, and FT3, FT4 and AMH gradually decrease along with the extension of the molding time are shown in a figure 9.

Serum ELisa results of 3.3.2BIW group mice

TABLE 17 serum ELisa results of BIW group mice

Note: the BIW model group was compared with the BIW control group, ^* P<0.05， ^** P<0.01; model group comparisons, to BIW1 group: ^Δ1 P<0.05， ^ΔΔ1 P<0.01; comparison with BIW2 group: ^Δ2 P<0.05， ^ΔΔ2 P<0.01; comparison with BIW3 group: ^Δ3 P<0.05， ^ΔΔ ³ P<0.01; comparison with BIW4 group: ^Δ4 P<0.05， ^ΔΔ4 P<0.01; comparison with BIW5 group: ^Δ5 P<0.05， ^ΔΔ5 P<0.01; comparison with BIW6 group: ^Δ6 P<0.05， ^ΔΔ6 P<0.01; comparison with BIW7 group: ^Δ7 P<0.05， ^ΔΔ7 P<0.01。

the serum ELisa results of the BIW group accord with normal distribution and homogeneity of variance, and LSD test in one-wayANOVA is adopted.

(1) TGAb: compared with the BIW control group, the TGAb concentrations of the BIW1 group and the BIW2 group are increased but the difference is not statistically significant (P is more than 0.05); the concentration of the Biw3 group and the Biw4 group is obviously increased (P < 0.05); the concentration of BIW5, biw6 and Biw7 groups is obviously increased (P < 0.01). Compared with the BIW model group, the concentration of the BIW5 group is obviously increased (P is less than 0.05) compared with that of the BIW1 group and the BIW2 group, the concentration of the Biw6 group and the Biw7 group is obviously increased (P is less than 0.01) compared with that of the BIW1 group and the BIW2 group, and the difference among the other groups has no statistical significance (P is more than 0.05).

(2) TPOAb: compared with the BIW control group, the concentration of the BIW1 group and the BIW2 group is increased, but the difference has no statistical significance (P is more than 0.05); the concentrations of TPOAb in the Biw3 group, the Biw4 group, the Biw5 group, the Biw6 group and the Biw7 group are obviously increased (P < 0.01). Compared with the BIW model group, the concentration of TPOAb in the Biw4 group, the Biw5 group, the Biw6 group and the Biw7 group is obviously higher than that in the BIW1 group and the BIW2 group (P is less than 0.01), the concentration of the BIW3 group is obviously higher than that in the BIW1 group (P is less than 0.01) and is obviously higher than that in the BIW2 group (P is less than 0.05), and the difference among the other groups has no statistical significance (P is more than 0.05).

(3) FT3, compared with the control group, the FT3 levels of the BIW1 group, the BIW2 group, the BIW3 group, the BIW4 group and the BIW5 group are reduced to some extent, and the difference does not have statistical significance (P is more than 0.05); FT3 levels significantly decreased in the BIW6 group (P < 0.01); FT3 levels were significantly reduced in the BIW7 group (P < 0.05). Compared with the BIW model group, the BIW6 group is obviously reduced compared with the BIW1 group and the BIW2 group (P is less than 0.01), and the difference among the rest groups has no statistical significance (P is more than 0.05).

(4) FT4: compared with a control group, the FT4 levels of the BIW1 group, the BIW2 group, the BIW3 group, the BIW4 group and the BIW5 group are reduced, and the difference has no statistical significance (P is more than 0.05); significantly decreased FT4 levels in the BIW6 group (P < 0.01); FT4 levels were significantly decreased in the BIW7 group (P < 0.05). Comparison between BIW model groups: differences between groups were not statistically significant (P > 0.05).

(5) TSH: compared with a control group, TSH levels of the BIW1 group, the BIW2 group, the BIW3 group and the BIW4 group are increased, and the difference has no statistical significance (P is more than 0.05); TSH levels were significantly elevated in the BIW5 group (P < 0.05); TSH levels were significantly elevated in BIW6 and BIW7 groups (P < 0.01). Comparison between BIW model groups: BIW7 group was significantly higher than BIW1 group (P < 0.01), significantly higher than BIW2 group (P < 0.05), BIW6 group was significantly higher than BIW1 group (P < 0.05), and the differences among the other groups were not statistically significant (P > 0.05).

(6) AMH: compared with a control group, the AMH levels of the BIW1 group and the BIW2 group are reduced, and the difference has no statistical significance (P is more than 0.05); AMH levels were significantly decreased in BIW3 group (P < 0.05); the AMH levels of BIW4, biw5, biw6 and Biw7 groups were significantly reduced (P < 0.01). Comparison between BIW model groups: BIW4, biw5, biw6 and Biw7 groups were significantly lower (P < 0.01) than BIW1 and BIW2 groups, and the differences among the other groups were not statistically significant (P > 0.05). The thyroid antibodies and TSH of mice in BIW group gradually increase with the molding time, and FT3, FT4 and AMH gradually decrease with the molding time as shown in figure 10.

3.4 determination of serum oxidative stress markers by enzyme-linked immunosorbent assay (ELISA)

The concentration of GSH-PX, MDA, ROS and SOD in serum of 3.4.1J group mice

TABLE 18 variation of the serum concentration of GSH-PX, MDA, ROS, and SOD in group J mice

Note: the J model group was compared with the J control group, ^* P<0.05， ^** P<0.01; comparison between model groups versus J1 group: ^Δ1 P<0.05， ^ΔΔ1 P<0.01; comparison with group J2: ^Δ2 P<0.05， ^ΔΔ2 P<0.01; comparison with group J3: ^Δ3 P<0.05， ^ΔΔ3 P<0.01; comparison with group J4: ^Δ4 P<0.05， ^ΔΔ4 P<0.01; comparison with group J5: ^Δ5 P<0.05， ^ΔΔ5 P<0.01; comparison with group J6: ^Δ6 P<0.05， ^ΔΔ ⁶ P<0.01; comparison with group J7: ^Δ7 P<0.05， ^ΔΔ7 P<0.01。

serum ELisa results in group J were normal distributed and uniform in variance, so the LSD test in one-wayaanova was used.

(1) GSH-PX: compared with the J control group, the concentration of GSH-PX in the J1 group and the J2 group is reduced, but the difference has no statistical significance (P is more than 0.05); group J3 concentrations were significantly reduced (P < 0.05); (ii) a The concentrations of the J4 group, the J5 group, the J6 group and the J7 group are obviously reduced (P < 0.01). J model group comparisons: group J5 was significantly lower (P < 0.05) than group J2; group J6 was significantly lower (P < 0.05) than group J1; the J6 group and the J7 group are obviously reduced compared with the J2 group (P < 0.01); the differences between the remaining groups were not statistically significant (P > 0.05).

(2) MDA: compared with the J control group, the MDA concentrations of the J1 group and the J2 group are increased but have no significant difference, and the statistical significance is not achieved (P is more than 0.05); the MDA concentration of J4 group and J5 group is obviously increased (P < 0.05); the MDA concentration of J6 group and J7 group is obviously increased (P < 0.01). Differences between model groups of J were not statistically significant (P > 0.05).

(3) ROS: compared with the J control group, the ROS concentrations of the J1 group, the J2 group and the J3 group are increased but have no significant difference, and the statistical significance is not achieved (P is more than 0.05); the J4 group ROS concentration was significantly increased (P < 0.05); ROS concentrations were significantly increased in J5, J6, and J7 groups (P < 0.01). Group J model group comparisons: the J4 group, the J5 group, the J6 group and the J7 group are all obviously increased (P < 0.01) compared with the J1 group; ROS concentration of J4 group and J5 group are obviously increased compared with that of J2 group (P < 0.05); the concentrations of the J6 group and the J7 group are obviously higher than those of the J2 group and the J3 group (P is less than 0.01), and the differences among the other groups have no statistical significance (P is more than 0.05).

(4) SOD (superoxide dismutase): compared with the J control group, the concentrations of the J1 group, the J2 group and the J3 group are reduced, but the difference has no statistical significance (P is more than 0.05); group J4 concentrations were significantly reduced (P < 0.05); the concentrations of the J5, J6 and J7 groups were significantly reduced (P < 0.01). Compared with the J model group, the J5 group is obviously reduced (P is less than 0.05) compared with the J1 group and the J2 group; the J6 group is obviously reduced compared with the J1 group (P < 0.01) and is obviously reduced compared with the J2 group (P < 0.05); the J7 group was significantly lower (P < 0.05) than the J1 group; the differences between the remaining groups were not statistically significant (P > 0.05). The J group oxidative stress markers ROS and MDA have a tendency of gradually increasing along with the extension of the molding time, and the activities SOD and GSH-PX of the antioxidase have a tendency of gradually decreasing along with the extension of the molding time, which is shown in figure 11.

The concentration of GSH-PX, MDA, ROS and SOD in serum of 3.4.2BIW group mice

TABLE 19 Change in serum GSH-PX, MDA, ROS, SOD concentration in BIW group mice

the serum oxidative stress markers of BIW group mice are normally distributed and have uniform variance, so the LSD test in one-way ANOVA is adopted.

(1) GSH-PX: compared with the BIW control group, the GSH-PX of the BIW1 group and the BIW2 group is reduced, but the difference has no statistical significance (P is more than 0.05); the GSH-PX of the Biw3 group and the Biw4 group are obviously reduced (P < 0.05), and the GSH-PX of the Biw5 group, the Biw6 group and the Biw7 group are obviously reduced (P < 0.01). Comparison between BIW model groups: the concentration of the Biw5 group and the Biw7 group is obviously lower than that of the BIW2 group (P < 0.05); the concentration of the Biw6 group is obviously lower than that of the BIW1 group (P < 0.01); the differences between the remaining groups were not statistically significant (P > 0.05).

(2) MDA: compared with the BIW control group, the MDA of the BIW1 group and the BIW2 group is increased but the difference is not statistically significant (P is more than 0.05); the MDA of the Biw3 group is obviously increased (P < 0.05), and the MDA of the BIW4 group, the Biw5 group, the Biw6 group and the Biw7 group is obviously increased (P < 0.01). . Comparison between BIW model groups: the concentration of BIW4 group, biw5 group, biw6 group and Biw7 group is obviously higher than that of BIW1 group (P < 0.05); the concentration of the Biw6 group and the Biw7 group is obviously higher than that of the BIW2 group (P < 0.01); BIW5 group was significantly higher than BIW2 group (P < 0.05); the differences between the remaining groups were not statistically significant (P > 0.05).

(3) ROS: compared with the BIW control group, the concentration of the BIW1 group and the BIW2 group is increased but the difference is not obvious, and the statistical significance is not achieved; ROS are obviously increased in Biw3 group and BIW4 group (P is less than 0.05); ROS were significantly elevated in the Biw5, biw6, biw7 groups (P < 0.01). Comparison between BIW model groups: the concentration of the Biw3 group, the BIW4 group, the Biw5 group, the Biw6 group and the Biw7 group is obviously higher than that of the BIW1 group (P is less than 0.01); the concentration of BIW6 group and BIW7 group is obviously higher than that of BIW2 group (P < 0.05); the concentration of BIW6 group is obviously higher than that of BIW3 group (P < 0.05); the differences between the remaining groups were not statistically significant (P > 0.05).

(4) SOD (superoxide dismutase): compared with the BIW control group, the SOD concentration of the BIW1 group, the BIW2 group and the BIW3 group is reduced, but the difference does not have statistical significance (P is more than 0.05); SOD concentration of Biw4 group and Biw5 group is obviously reduced (P is less than 0.05); SOD concentration of Biw6 group and Biw7 group is obviously reduced (P is less than 0.01). Comparison between BIW model groups: the concentration of the Biw6 group and the Biw7 group is obviously lower than that of the BIW1 group (P < 0.05); the concentration of the Biw7 group is obviously lower than that of the BIW2 group (P < 0.05); the differences between the remaining groups were not statistically significant (P > 0.05). The oxidative stress markers ROS and MDA have a tendency of gradually increasing with the molding time, and the activities SOD and GSH-PX of antioxidase have a tendency of gradually decreasing with the molding time, as shown in FIG. 12.

4 model group J and BIW form model condition summary

TABLE 20J and BIW model summary

Note: oxidative Stress (OS); ≈ indicates a marked/significant increase compared to the control group (P <0.05/P < 0.01); ↓: indicating a significant/significant reduction compared to the control group (P <0.05/P < 0.01).

4.1 Experimental Autoimmune Thyroiditis (EAT) mouse model conditions

(1) J model group: the J model has no statistical significance in thyroid antibodies (TGAb, TPOAb), first work, follicle count at each stage, corpus luteum difference from the J control group of mice in J1 group and J2 group of mice at 7-9 weeks of model building; in the J3 group at 11 weeks of model making, 3 mice have thyroid lymphocyte infiltration and follicular structure change, thyroid antibodies (TGAb and TPOAb) of the J3 group are increased, namely 60% of mice become EAT models, the number of corpus luteum is obviously reduced compared with that of the J control group, and the difference of follicle count at each level has no statistical significance; thyroid lymphocyte infiltration, follicular structure change and thyroid antibody (TGAb and TPOAb) obvious increase appear in J4 group mice at 13 weeks of model building, namely the model forming rate of the EAT model is 100%, at the moment, primordial follicles, primary follicles and secondary follicles of the mice are obviously/obviously reduced compared with a J control group, and atretic follicles are obviously/obviously increased; the trend is kept in 15-19 weeks of the subsequent molding, which indicates that the hypofunction of ovarian reserve begins to appear in the J4 group of the J model group in 13 weeks of the molding; hypothyroidism appeared in 17-and 19-week-old J6 and J7 EAT mice, which is a stage of hypothyroidism in which EAT combined with a decline in ovarian reserve function.

(2) BIW model group: the BIW model is formed in BIW1 mice of 7 weeks of molding, only the (6) mice have slightly changed thyroid follicular structure, thyroid lymphocyte infiltration, thyroid antibody (TGAb, TPOAb), jiagong, follicle count at each level, and corpus luteum and J control group difference have no statistical significance; 3/6 mice in BIW2 group at 9 weeks of molding have thyroid lymphocyte infiltration and follicular structure change, thyroid antibodies (TGAb and TPOAb) in BIW2 group are increased, namely 50% of mice become EAT model, at this time, the corpus luteum number is obviously reduced compared with BIW control group, but the difference of follicle count at each level has no statistical significance; thyroid lymphocyte infiltration, follicular structure change and thyroid antibodies (TGAb and TPOAb) are obviously increased in BIW3 mice at 11 weeks of molding, the molding rate of an EAT model is 100%, and at the moment, primary follicles, secondary follicles of the mice are all reduced compared with a BIW control group, and atretic follicles are increased; the trend is kept in 13-19 weeks of the subsequent molding, which indicates that the BIW model group begins to have low ovarian reserve function in the BIW3 group in 11 weeks of the molding; the EAT mice in BIW5 group at 15 weeks of molding have obviously increased TSH level compared with the control group, but the FT3 and FT4 levels are reduced but the difference has no statistical significance, which indicates that subclinical hypothyroidism appears at the stage; the EAT mice of BIW6 group and BIW7 group at 17 weeks, 19 weeks of model building have obviously increased TSH level and obviously decreased FT3 and FT4 level, which indicates that hypothyroidism and ovary reserve function decline appear at the stage.

(3) Comparing the J model group with the BIW model group: the time when EAT mice in BIW model group begin to have ovarian reserve hypofunction is 11 weeks of modeling, and the time when EAT mice in J model group begin to have ovarian reserve hypofunction is 13 weeks of modeling. The modeling time of Kunming mice with the decreased ovarian reserve function caused by the EAT with normal first work in the J model group is 13-15 weeks, and the modeling time of Kunming mice with the decreased ovarian reserve function caused by the EAT with normal first work in the BIW model group is 11-13 weeks. The high iodine water is continuously fed for molding for 17-19 weeks, and the J6 group, the J7 group, the BIW6 group and the BIW7 group are DOR models caused by hypothyroidism EAT. The BIW model group at 11-19 weeks of model building and the J model group at 13-19 weeks of model building all had fewer primordial follicles, primary follicles, secondary follicles, and corpus luteum than the control group, and had more atretic follicles. The reason why the mature follicles among the groups are not different is that the material drawing time is in the prophase of estrus (lasting 9-18 hours), the follicles are in the growth and development stage, most follicles in each group are not mature, the corpus luteum of the mouse is maintained for 3.70 +/-0.34 days [2], the corpus luteum in the previous period of the prophase of the estrus of the mouse is not completely regressed, and the corpus luteum can still be seen.

4.2 model selection for Normal first-order-onset ovarian reserve function decline in EAT mice

TABLE 21 overview of EAT model group for normal first work inducing ovarian reserve functional decline

Note: EAT model group of normal afunction, J4 group, J5 group compared to J control group: ^* P<0.05， ^** P<0.01; BIW3 and BIW4 groups of the EAT model group of normal A function were compared with the BIW control group: ^# P<0.05， ^## P<0.01; comparison between groups of EAT models of normal first function: in comparison with the group J4, ^Δ4 P<0.05， ^ΔΔ4 P<0.01; in comparison with the group J5, ^Δ5 P<0.05， ^ΔΔ5 P<0.01; compared with the BIW3 group, ^▲3 P<0.05， ^▲▲3 P<0.01; in contrast to the BIW4, ^▲4 P<0.05， ^▲▲4 P<0.01。

(1) Thyroid lymphocyte infiltration strength, thyroid follicular structure change, thyroid antibody level, primordial follicles, atretic follicles and AMH of normal A EAT model groups (BIW 3 group, J4 group, BIW4 group and J5 group) are compared among groups, and the groups are uniform in variance and are tested by adopting one-factor variance LSD, and the result shows that: thyroid lymphocyte infiltration intensity, thyroid follicular structural changes, thyroid antibody levels, primordial follicles, atretic follicles, AMH differences among BIW3, J4, BIW4, J5 groups were not statistically significant.

(2) Although the differences among the EAT model groups with normal A function have no statistical significance, the BIW4 group with 13 weeks of modeling has significantly reduced primordial follicles and AMH (P < 0.05), and the thyroid follicle structural change and TPOAb antibody level are significantly increased (P < 0.05) compared with the control group, so that the EAT model group with normal A function has the characteristics of relatively early modeling, thyroid destruction and obvious ovarian reserve function reduction, and can be used as a modeling reference for subsequent experimental study.

4.3 Instructions that the control group (7 weeks old) can be used for control

Studies have suggested that middle-aged rats in nature exhibit an irregular estrous cycle from 9-12 months of age, and that natural decline in ovarian function begins to occur, usually characterized by an extended estrous cycle. Mice did not reach the natural decline stage during the molding period of the study at about 4-7 months of age (15 weeks of age-28 weeks of age). For further strict observation, there were observed three additional 3 groups of Kunming female mice of different ages without any treatment, which were 16 weeks old (W16 group corresponding to molding 7 weeks), 20 weeks old (W20 group corresponding to molding 11 weeks), and 24 weeks old (W24 group corresponding to molding 15 weeks). The results of the serum AMH of 3 groups of mice were as follows:

TABLE 22 variation of AMH concentration in serum of group 3 mice (X. + -. SD)

Note: and W ₁₆ Compared with the group, the group comparison method has the advantages that, ^* P<0.05， ^** P<0.01; and W ₂₀ Compared with the group, the group comparison method has the advantages that, ^# P<0.05， ^## P<0.01; and W ₂₄ Compared with the group, the following steps are carried out, ^Δ P<0.05， ^ΔΔ P<0.01

the statistical result shows that the AMH concentration difference in the serum of each group of mice has no statistical significance (P is more than 0.05), so that the J control group and the BIW control group at the age of 16 weeks can be compared with each group of the model when the model is made and taken in the experiment.

4.5, summary

(1) J model group and BIW model group: the thyroid lymphocyte infiltration intensity score, the thyroid follicular structure change score, the thyroid antibody (TGAb, TPOAb) level, TSH and atretic follicles all tended to increase, while AMH, FT3, FT4, primordial follicles, primary follicles, secondary follicles and corpus luteum all tended to decrease with the increase of the molding time in the J model group and the BIW model group. The BIW model group and the J model group respectively show thyroid lymphocyte infiltration intensity scores, thyroid follicular structure change scores, thyroid antibody (TGAb and TPOAb) levels, oxidative stress markers (ROS and MDA) levels and atretic follicles from 11 weeks and 13 weeks of model building, which are all obviously higher than those of a control group, and primordial follicles, primary follicles, secondary follicles, corpus luteum and antioxidant markers (SOD and GSH-PX) levels are all obviously lower than those of the control group. The BIW model group (BIW 3-BIW7 group) of 11 weeks to 19 weeks of model building and the J model group (J4-J7 group) of 13 weeks to 19 weeks of model building have autoimmune thyroiditis and low ovarian reserve function. Hypothyroidism appeared in the J6, J7 and BIW6, BIW7 groups in the 17-19 week group on high iodine water feeding.

(2) Time to ovarian hypofunction in Experimental Autoimmune Thyroiditis (EAT) mice: the BIW model group is earlier than the J model group, namely the antigen immune induction of 1 week and 2 times has better modeling time than that of 1 week and 1 time;

(3) EAT mice with normal A function, combined with low ovarian reserve function, have no statistical significance in thyroid lymphocyte infiltration strength, thyroid follicular structure change, thyroid antibody level, primordial follicles, atretic follicles and AMH difference among BIW3, BIW4, J4 and J5 groups.

5. Conclusion

Experimental Autoimmune Thyroiditis (EAT) has a negative impact on ovarian reserve function and can induce the DOR model by EAT. High iodine water (0.64 g.L) ^-1 ) Feeding combined with 2 antigen immune inducements per week (500 mg. L) ^-1 ) DOR caused by EAT with normal onychosis can be established in 11-13 weeks, and DOR caused by EAT with hypothyroidism can be established in more than 15 weeks. High iodine water (0.64 g.L) ^-1 ) Feeding combined with 1 antigen immune induction per week (500 mg. L) ^-1 ) DOR caused by EAT with normal onycholysis can be established in 13-15 weeks, and DOR caused by EAT with hypothyroidism can be established in more than 17 weeks. Antigen immunity induction matched with high iodine water feeding is carried out 2 times per week, the modeling of the EAT merged DOR model with normal thyropathy is relatively early, the damage to the thyroid gland is obvious, and the ovarian reserve is obviously reduced.

Claims

1. A method for constructing an animal model with low ovarian reserve function induced by autoimmune thyroiditis is characterized by comprising the following steps: the method comprises the following steps:

taking animals, injecting a primary immune emulsifier subcutaneously for 2 weeks continuously, 1-2 times per week, and injecting a reinforced immune emulsifier for 5-13 weeks from the 3 rd week, 1-2 times per week;

feeding said animal daily with a sodium iodide solution during the injection of the emulsifier;

the primary immune emulsifier is prepared by mixing a pig thyroglobulin antigen solution with Freund's complete adjuvantThe concentration of the antigen of the finished pig thyroglobulin is 200-800 mg.L ^-1 The emulsion of (4);

2. The construction method according to claim 1, characterized in that: the time of injection is monday and thursday of the week, preferably the time of injection is started at 9 am on monday and thursday of the week.

3. The construction method according to claim 1, characterized in that: the animal is a mammal, preferably a mouse; the mouse is Kunming mouse, preferably Kunming mouse with the age of 4-7 months.

4. The construction method according to claim 1, characterized in that: the injection site is the neck, back, inner thigh and/or abdomen.

5. The construction method according to claim 1, characterized in that: the injection of the immune-enhancing emulsifier is performed for 5 weeks, 7 weeks, 9 weeks, 11 weeks or 13 weeks, preferably 11 weeks.

6. The construction method according to claim 1, characterized in that: the injection of the immune-enhancing emulsifier is carried out for 13 weeks, and then the feeding of the sodium iodide solution is continued until 17-19 weeks.

7. The construction method according to claim 1 or 6, characterized in that: the concentration of the sodium iodide solution is 0.3-1 g.L ^-1 。

8. The construction method according to claim 1, characterized in that: the preparation method of the primary immune emulsifier comprises the following steps:

freund's complete adjuvant and porcine thyroglobulin antigen solution were mixed at 1:1 volume ratio of the mixture, at a rotation speed of 1000-5000 r/min to form a viscous emulsifier, wherein the concentration of the porcine thyroglobulin antigen is 500 mg.L ^-1 。

9. The construction method according to claim 1, characterized in that: the preparation method of the immunity enhancing emulsifier comprises the following steps:

freund's incomplete adjuvant and porcine thyroglobulin antigen solution were mixed at a ratio of 1:1 volume ratio, and mixing at 1000-5000 r/min to obtain viscous emulsifier with porcine thyroglobulin antigen concentration of 500 mg.L ^-1 ；

The swine thyroglobulin antigen solution is prepared by dissolving a swine thyroglobulin antigen in a phosphate buffer solution, and the concentration of the swine thyroglobulin antigen solution is 0.5-1 mg/ml.

10. An autoimmune thyroiditis-induced hypoovarian reserve function animal model, characterized in that: it is a mouse model constructed according to the method described above.