Construction method of thyroid-associated ophthalmopathy animal model
By using the method of homogenate preparation and injection modeling, Freund's adjuvant was used to induce an autoimmune response in mice, which solved the problems of the complexity and long cycle of the existing models and established an animal model of thyroid-related eye disease that conforms to the pathological characteristics of human TAO.
Patent Information
- Application Number
- CN202510819948.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-19
- Publication Date
- 2025-09-19
AI Technical Summary
The existing methods for constructing animal models of thyroid-related eye diseases are complex, have long cycles, low repetition rates, and are quite different from the pathogenesis of the disease, and cannot completely replicate the pathological characteristics of human TAO.
The homogenate preparation and injection modeling method was used. The soft tissue behind the mouse eye was extracted and mixed with Freund's adjuvant as an emulsifier, and multi-point injection was performed to induce an autoimmune response in mice and simulate the TAO disease process.
A more comprehensive and realistic animal model of TAO pathological characteristics has been established. It is easy to operate and low-cost, and its pathological characteristics are consistent with human TAO, including changes in eye and thyroid tissues.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of animal models, and in particular to a method for constructing an animal model of thyroid-related eye disease. Background Art
[0002] Thyroid associated ophthalmopathy (TAO), also known as Graves' ophthalmopathy (GO), is an autoimmune disease that ranks first in the incidence of orbital diseases in adults. Its main manifestations are upper eyelid retraction, edema, orbital tissue erythema and proptosis, which can further develop into blindness.
[0003] The pathogenesis of TAO is complex and involves multiple factors, including genetic, immune, and environmental factors. Its primary pathogenesis involves the body's misrecognition of target cell surface molecules through cross-antigen reactions between thyroid and orbital tissues, loss of target organ immune tolerance, and overactivation of local immunity. In TAO, the thyroid-stimulating hormone receptor (TSHR) is the primary autoantigen, also expressed in orbital fibroblasts and adipocytes. TSHR antibodies, activated T cells, and macrophages play a crucial role in the pathogenesis of TAO by activating TSHR in orbital fibroblasts and adipocytes, initiating cell proliferation, and initiating orbital inflammation.
[0004] Since orbital tissue biopsy is difficult in clinical practice, establishing an ideal animal model is of great significance for the development of new clinical therapies for TAO. To date, TAO animal modeling methods are mainly based on inducing experimental animals to produce anti-thyroid stimulating hormone receptor antibodies (TRAbs) and then recruiting autoimmune T lymphocytes. The existing modeling methods mainly include: (1) TSHR immune model, which simulates the autoimmune response of TAO by injecting immunized animals with antigens related to the thyroid-stimulating hormone receptor (TSHR), but cannot completely replicate all the characteristics of human TAO; (2) TSHR nucleic acid immune model, which directly uses the nucleic acid sequence of TSHR to immunize animals to induce a stronger immune response, which may cause excessive immune response, resulting in pathological changes in the model that are different from human TAO; (3) TSHR transfected cell model, which can produce TSHR in the animal body by transfecting the TSHR gene into the animal's cells, thereby simulating the pathological process of TAO, but the transfection efficiency and expression level may affect the reliability and repeatability of the model; (4) orbital fibroblast (OFs) model, which can help understand the process of extraocular muscle fibrosis, but a single cell model may not fully reflect the multifactorial pathological process of TAO.
[0005] These animal models provide powerful tools for exploring the intrinsic relationship between local and systemic immune microenvironmental disorders in the TAO orbit, promoting the development of new drugs. However, existing TAO modeling methods still have shortcomings such as complex modeling, long modeling cycles, low reproducibility, and significant differences in the pathogenesis of TAO diseases. Summary of the Invention
[0006] Technical problems solved by the present invention:
[0007] Among current modeling methods, the most commonly used methods are hTSHR-A subunit plasmid electroporation and hTSHR-A subunit adenovirus transfection. However, existing TAO modeling methods still have the disadvantages of being relatively complex, having a long modeling cycle (11-37 weeks), and existing TAO models only utilize TSHR to trigger TAO, which is obviously imperfect and has significant differences from the pathogenesis of TAO. The construction process of the present invention uses homogenate preparation and injection modeling, which can effectively replicate the pathogenesis of TAO and is easy, fast, and low-cost to operate.
[0008] The technical solution adopted in the present invention is:
[0009] Based on the foregoing, the present invention provides a method for constructing an animal model of thyroid-related eye disease.
[0010] To expand on this, it means:
[0011] A method for constructing an animal model of thyroid-associated eye disease comprises extracting a homogenate of the posterior soft tissue of mice, mixing the homogenate with complete Freund's adjuvant to form an emulsifier, and injecting the emulsifier into the mice at multiple points to induce an autoimmune response in the mice, thereby producing a mouse model of thyroid-associated eye disease.
[0012] According to some preferred embodiments, the posterior ocular soft tissue is the soft tissue obtained by removing the eyeball and the optic nerve and taking the remaining muscle and fat tissue.
[0013] According to some preferred embodiments, multiple injection sites include intraorbital and subcutaneous injection (at the eyelid) of the mouse.
[0014] According to some preferred embodiments, the method for preparing the posterior ocular soft tissue homogenate is as follows: the posterior ocular soft tissue is homogenized in PBS (PBS concentration is 1×), and centrifuged at 0-40°C and 500-10000g for 5-30 minutes, the supernatant is taken, and the supernatant is ultracentrifuged at 20000g for 0.5-3 hours, and the supernatant is aspirated to obtain the posterior ocular soft tissue homogenate.
[0015] According to some preferred embodiments, the volume ratio of the posterior ocular soft tissue homogenate to complete Freund's adjuvant is 1:0.5-5.
[0016] According to some preferred embodiments, the injection is performed 1 to 7 times per week from week 1 to week 5. The injection dosage of the emulsifier is 0.2 ml per animal.
[0017] According to some preferred embodiments, the adjuvant emulsified with incomplete Freund's adjuvant is injected from week 3 to week 6 for 1 to 5 consecutive weeks, 1 to 7 times per week. The dilution volume of incomplete Freund's adjuvant is 0.5 to 5 times. The injection volume of the adjuvant emulsified with incomplete Freund's adjuvant is 0.2 ml per mouse. The injection dose is 0.1 ml per eye per mouse, calculated as 0.0009 ml / g body weight.
[0018] According to some preferred embodiments, the rodents may be mice or rats.
[0019] Second, the present invention provides an application of a model constructed by the aforementioned construction method in a drug screening test.
[0020] According to some preferred embodiments, the application is a test for screening drugs for treating autoimmune thyroid disease.
[0021] The beneficial effects achieved by the present invention are:
[0022] The present invention starts from the pathological mechanism of autoimmune diseases. By extracting orbital tissue from mice, the immune system is repeatedly stimulated with the assistance of Freund's adjuvant, causing the immune system to misidentify the orbital tissue, completely simulating the occurrence process of TAO disease, and thus establishing a more comprehensive and closer to the real TAO pathological characteristics autoimmune thyroid-related eye disease model.
[0023] The construction process of the present invention uses homogenate preparation and injection modeling, which is convenient, reduces modeling time, and is low-cost. Existing electroporation and adenovirus transfection technologies, on the other hand, have complex processes and require high operator skills, while also carrying a significant risk of inducing other immune reactions.
[0024] The construction process of the present invention uses complete Freund's adjuvant for a primary immunization to stimulate a strong immune response, followed by a booster immunization with incomplete Freund's adjuvant to maintain immune memory. After the initial immunization, the body has already established a certain level of immune memory cells. Subsequent booster immunizations with incomplete Freund's adjuvant can, on the one hand, re-stimulate immune memory cells, causing them to proliferate and differentiate, maintaining a high level of immune response and prolonging immune memory. On the other hand, due to its relatively mild immunostimulatory properties, it can reduce the side effects of excessive immune responses, such as excessive local inflammation and tissue damage, thereby making the immune response more sustained and stable.
[0025] The pathological characteristics of the animal model constructed by the present invention are more consistent with clinical pathological characteristics: that is, the pathogenesis of TAO disease is completely replicated, and the ocular pathological characteristics are obvious. The changes in TSH, TRAb, FT3, and FT4 in serum and the pathological changes in ocular soft tissue and thyroid tissue are consistent with the pathological changes of thyroid-related eye disease. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 Schematic diagram for constructing an animal model of thyroid-related eye disease;
[0027] Figure 2 This is the eye observation result of rats one week after injection;
[0028] Figure 3 Figure 3 is the weight growth curve of rats after modeling;
[0029] Figure 4 This is the result diagram of the ratio of food intake to body weight of rats after modeling;
[0030] Figure 5 The results of ELISA detection of the expression levels of FT3, FT4, TSH and TRAb in serum;
[0031] Figure 6 This is the result of HE staining of rat thyroid (magnification 4 times);
[0032] Figure 7 This is the result of HE staining of rat thyroid (magnification 20 times);
[0033] Figure 8 This is the HE staining result of the soft tissue behind the eye of rat (magnification 10 times);
[0034] Figure 9 Figure 1 is the result of immunohistochemical staining of rats (magnification 4 times);
[0035] Figure 10 This is a photo of the gross specimens of rat eye tissue and thyroid gland;
[0036] Figure 11 This is a graph showing the ratio of rat thyroid mass to eyeball / posterior eye fat. DETAILED DESCRIPTION
[0037] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.
[0038] Example 1 - Preparation of posterior ocular soft tissue homogenate
[0039] Materials: The eyeballs and optic nerves of SD rats were removed, and the remaining muscle and fat tissue were obtained;
[0040] Homogenize: Weigh and wash with cold saline; homogenize the removed orbital tissue in PBS (mass ratio of 1:2) and centrifuge at 500-10,000g for 5-30 minutes at 4°C. Ultracentrifuge the supernatant at 20,000g for 0.5-3 hours. Aspirate the supernatant to obtain a soft tissue homogenate. Homogenize the removed orbital tissue in PBS and centrifuge at 1,000g for 10 minutes at 4°C. Ultracentrifuge the supernatant at 10,000g for 1 hour. Aspirate the supernatant. In the above, care should be taken to prepare and use the product immediately to avoid repeated freezing and thawing.
[0041] Example 2 - Construction of TAO Animal Model
[0042] For 5 consecutive weeks, the patients were injected with posterior ocular soft tissue homogenate and Freund's adjuvant emulsion (volume ratio of 1:1, 1:2) and posterior ocular soft tissue homogenate and incomplete Freund's adjuvant emulsion (volume ratio of 1:1, 1:2). Diet, body weight and ocular condition were recorded every week. Figure 1 shown.
[0043] Every 1-2 weeks, the orbital homogenate was emulsified with complete Freund's adjuvant in multiple doses. The injection frequency was once per week, with 0.1 ml injected into each eye of each mouse, which was 0.0009 ml / g based on body weight.
[0044] Inject an adjuvant prepared by emulsifying the orbital homogenate with incomplete Freund's adjuvant once a week for 3-5 weeks. Each mouse is injected with 0.1 ml per eye, which is 0.0009 ml / g based on body weight.
[0045] 1. Ocular Observation of Thyroid-Related Eye Disease Animal Model
[0046] like Figure 2 As shown, compared with the PBS group and the PBS + Freund's adjuvant group (volume ratio 1:1), the rats injected with Freund's adjuvant and posterior soft tissue homogenate (emulsified preparations at volume ratios of 1:1 and 2:1) showed symptoms such as proptosis, congestion and edema, corneal exposure, and eyelid retraction. Among them, the eye symptoms of the model group with 1:1 emulsification were more obvious.
[0047] 2. Changes in the ratio of body weight to fed body weight in animal models of thyroid-related eye disease
[0048] The changes in rat body weight Figure 3 As shown in the figure, the weight gain of the PBS group was greater than that of the control group, and the model group (1:1) and PBS+Freund's adjuvant group showed weight loss at 5 weeks after modeling.
[0049] Food intake weight Figure 4 As shown, at week 5, the model group (1:1) had the lowest food intake but less weight gain compared with the control group.
[0050] 3. Serological testing
[0051] (1) Preparation of serum: The rats were killed and blood was quickly drawn from the heart before the blood coagulated. The specific operation was as follows: the rats were fixed on a foam board in a supine position and placed horizontally. After the skin was disinfected, the thumb and index finger of the left hand gently touched the rat's heart to more easily find the heartbeat. A 1 ml syringe was held in the right hand and the strongest heartbeat was selected. After piercing the heart, blood automatically entered the syringe. The amount of blood collected each time was about 0.5-0.8 ml. After blood collection, the samples were allowed to stand at room temperature for 1-2 hours, centrifuged at 4°C, 3000 rpm, for 10 minutes, and the upper layer of serum was separated using a micropipette mortar. The average amount of serum was 0.20-0.35 ml. The samples were labeled according to different groups, divided as needed, and stored in a -80°C refrigerator for inspection to avoid repeated freezing and thawing. The samples were thawed at 4°C before use to ensure that the samples were evenly and fully thawed.
[0052] (2) Detection of thyroid function: abnormal free triiodothyronine (FT3), free thyroxine (FT4), serum thyroid stimulating hormone (TSH) or elevated levels of thyroid-related antibodies, namely thyroid stimulating hormone receptor antibodies (TRAb).
[0053] (3) ELISA test: Take out the ELISA kit, equilibrate at room temperature for 30 minutes, dilute with distilled water 1:20, and mix well. Take out the enzyme-labeled plate, set up one blank control well at random, and set up 2 wells for each calibrator. No liquid is added to the blank control well, and 50 μL of the corresponding calibrator is added to each well of the calibrator. 50 μL of the serum to be tested is added to each of the remaining test wells. 50 μL of biotin labeling solution is added to all wells except the blank well, mixed well, covered with a sealing film, and incubated at 37°C for 60 minutes. Wash the plate manually 3 times, let it stand for 10 seconds each time, pat dry, wash the plate 3 times with a plate washer, and pat dry. Add 50 μL of enzyme-labeled avidin to all wells except the blank well, mix well, cover with a sealing film, and incubate at 37°C for 30 minutes. Repeat the above washing. Add 50 μL of color developer A and B to each well, mix well, and incubate at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well. Read the results using an enzyme-labeled instrument at a wavelength of 450 nm. Use a blank well to adjust the zero point first, then measure the optical density (OD) of each well. Draw a standard curve and convert the concentration of the corresponding samples.
[0054] ①ELISA detection of FT3 and FT4, such as Figure 5As shown in the figure, the modeling group was elevated compared with the control group, which was consistent with the pathological changes of TAO.
[0055] ②ELISA test for TSH, such as Figure 5 As shown in the figure, the modeling group was lower than the control group, which was consistent with the pathological changes of TAO.
[0056] ③ELISA detection of TRAb, such as Figure 5 As shown, the modeling group was elevated compared with the control group, which was consistent with the pathological changes of TAO;
[0057] The foregoing Figure 5 Medium model group (1:1).
[0058] 4. Pathological examination
[0059] (1) Rat thyroid tissue sections and HE staining, such as Figure 6-7 HE staining of thyroid tissue showed that compared with the control group, the TAO model rats had a large number of lymphocyte infiltration in the thyroid gland, some thyroid follicles were hyperplastic and enlarged, the colloid in the thyroid follicles was thin, and the follicular epithelial cells were hyperplastic, which was consistent with the pathological changes of TAO.
[0060] (2) Rat orbital soft tissue sections and HE and immunohistochemical staining, such as Figure 8-9 Compared with the PBS control group, TAO model rats showed thickening of the extraocular muscle fibers and lymphocyte infiltration; increased retrobulbar adipocytes and lymphocyte infiltration; and increased retrobulbar hyaluronic acid deposition and lymphocyte infiltration. These changes are consistent with the pathological manifestations of TAO.
[0061] (3) Observation of rat thyroid and eye tissue dissection
[0062] like Figure 10 As shown, the model group had increased soft tissue behind the eye and enlarged thyroid gland.
[0063] like Figure 11 As shown in the figure, the ratio of eyeball to posterior eye fat mass in the modeling group was less than that in the control group, and the posterior eye fat increased in the modeling group; Figure 10-11 in the model group (1:1).
[0064] In summary, the above changes are consistent with the pathological manifestations of TAO.
Claims
1. A method for constructing an animal model of thyroid-associated eye disease, comprising extracting a homogenate of the posterior soft tissue of mice, mixing the homogenate with complete Freund's adjuvant to form an emulsifier, and injecting the emulsifier into mice at multiple points to induce an autoimmune response in the mice, thereby producing a mouse model of thyroid-associated eye disease.
2. The construction method according to claim 1, characterized in that The soft tissue behind the eye is the soft tissue obtained by removing the eyeball and optic nerve and taking the remaining muscle and fat tissue.
3. The construction method according to claim 1, characterized in that Multiple injection sites included intraorbital and subcutaneous injections in mice.
4. The construction method according to claim 3, characterized in that From week 1 to week 5, inject 1 to 7 times per week.
5. The construction method according to claim 1, characterized in that The preparation method of the posterior ocular soft tissue homogenate is as follows: the posterior ocular soft tissue is homogenized in PBS, and centrifuged at 0-40°C and 500-10,000g for 5-30 minutes, the supernatant is collected, and the supernatant is ultracentrifuged at 20,000g for 0.5-3 hours, and the supernatant is aspirated to obtain the posterior ocular soft tissue homogenate.
6. The construction method according to claim 1, characterized in that The volume ratio of posterior ocular soft tissue homogenate to complete Freund's adjuvant is 1:0.5-5.
7. The construction method according to claim 1, characterized in that Rodents are rats or mice.
8. Use of a model constructed by the construction method according to any one of claims 1 to 7 in a drug screening test.
9. The use according to claim 8, characterized in that The application is a trial to screen for drugs to treat autoimmune thyroid disease.