动物造模,药效评价,甲状腺功能亢进症 z-bio 2024-08-01 919

　　（1） Exogenous Thyroxine Supplementation Method

　　The modeling mechanism involves artificially increasing the levels of thyroid hormone in animals through oral or injection administration, resulting in various symptoms of hyperthyroidism.

　　[Method of Modeling]

　　1. Kunming mice weighing 18-20g were orally administered with a suspension of 320mg/kg thyroid tablets for 9 consecutive days.

　　2. Adult rats weighing 180-220g were orally administered with a suspension of thyroid tablets at a dose of 40mg/d for 7 consecutive days. Or administer levothyroxine sodium 1mg/(kg · d) by gavage to body weight for 10 consecutive days.

　　3. New Zealand rabbits weighing 2.0-2.5kg were subcutaneously injected with 0.5mg/(kg · d) of levothyroxine sodium for 12-14 days.

　　After modeling various animals, food intake, water intake, body temperature, electrocardiogram, and serum T3 and T4 levels can be measured.

　　【 Model Characteristics 】 After administering exogenous thyroid hormone, animals may experience increased food and water intake, elevated body temperature, faster heart rate, and increased serum T3 and T4 levels. The rabbit's heart is enlarged, systolic blood pressure increases, and plasma atrial natriuretic peptide secretion increases. These clinical manifestations are very similar to the symptoms of hyperthyroidism in humans.

　　The exogenous thyroxine supplementation method induced hyperthyroidism model is suitable for studying the biological effects of excessive thyroxine on the body, as well as screening and pharmacological studies of anti hyperthyroidism drugs. This model involves directly administering exogenous thyroid hormone to animals, causing high levels of thyroid hormone in their bodies and resulting in a series of clinical manifestations. Therefore, the dosage of medication is closely related to the type and modeling time of the model. Excessive dosage can shorten the modeling time, but may increase the mortality rate. However, the increase of exogenous thyroid hormone feedback inhibits the secretion of thyroid stimulating hormone (TSH) in the pituitary gland, suppressing the secretion of thyroid gland, which is inconsistent with the pathogenesis and pathological changes of hyperthyroidism. This large mouse model can be used for studying the biological effects of thyroid hormone and the pharmacological effects of general drugs for treating hyperthyroidism.

　　Rabbits are susceptible animals to thyroid hormones. The blood pressure of hyperthyroidism induced hypertension established with exogenous thyroxine from rabbits is stable, and during the modeling process of hyperthyroidism induced hypertension, heart rate, systolic blood pressure, and plasma ANP levels are positively correlated with cardiac mass index.

　　（2） Immune induced goiter

　　Thyroid enlargement (GD) is an autoimmune disease in which TSHR specific antibodies (TRAb) are present in the patient's serum, which bind to TSHR and cause sustained secretion of thyroid hormone, making it the most important factor in causing hyperthyroidism. Immunizing animals with fibroblasts expressing human TSHR or expression vectors expressing human TSHR cDNA can induce the production of specific antibodies TRAb against TSHR, which can lead to clinical manifestations similar to human GD.

　　[Method of Modeling]

　　1. Cellular immunity: 1 × 10000000 mouse fibroblasts transfected with hTSHR cDNA, RT4.15HP, were injected into 7-week-old female AKR/N (H-2k) mice and immunized once every 2 weeks for a total of 6 immunizations. Two weeks after the last immunization, serum thyroid hormone levels and TSH stimulating antibodies (TSAb) were measured, as well as histopathological examination of the tissue.

　　2. The constructed hTSHR pcDNA100 μ g plasmid vector was used for nucleic acid immunization, and intramuscular injection immunization was performed on 6-8 week old BALB/c female mice at weeks 1, 4, 7, 9, and 10. Blood samples were collected from the eye sockets at weeks 6 and 11 to detect serum levels of T4, TRAb, TSAb, and TSH receptor blocking antibody (TBAb); The mice were euthanized in the 11th week, and the thyroid gland was taken for histopathological examination.

　　3. Adenovirus immunization: The adenovirus vector encoding the full-length hTSHR will be injected intramuscularly into 6-week-old BALB/c mice, immunized once every 3 weeks for a total of 3 immunizations. Eight weeks after the last immunization, all mice were euthanized and serum levels of T4 and TSAb were measured, as well as pathological examination of thyroid and eye tissues.

　　[Model Features] The above models all use vectors expressing hTSHR to inoculate animals, in order to stimulate their immune system to produce autoantibodies against TSHR. Modeling requires multiple immunizations to produce active antibodies, but not all animals can induce hyperthyroidism symptoms.

　　About 25% of the mice in the cellular immune experimental group developed hyperthyroidism: increased TSH binding inhibitory immunoglobulin (TBHs) activity, positive TSAb, elevated T4 levels, diffuse thyroid enlargement, and thyroid cell proliferation.

　　The levels of T4, TRAb, and TSAb in the nucleic acid immunization group significantly increased from the 6th week, and the thyroid gland enlarged, showing signs of hyperthyroidism such as the generation of numerous new follicles, proliferation of follicular epithelial cells, and glial condensation. After intramuscular injection of adenovirus vector encoding full-length hTSHR (AdCMVTSHR) into immunized mice, 55% of female and 33% of male BALB/c mice showed symptoms of hyperthyroidism, elevated T4 levels, and increased thyroid stimulating immunoglobulin (TSI) antibodies after 8 weeks.

　　Due to the fact that most hyperthyroidism is caused by organ specific autoimmunity, animals are immunized with cells or plasmid vectors expressing hTSHR based on the etiology to stimulate their immune system and produce specific antibodies against TSHR, thereby inducing animal models similar to human GD.

　　There are three types of TRAb in the human body, namely TSH stimulating antibody (TSAb), TSH stimulating blocking antibody (TBAb), and thyroid growth immunoglobulin (TGI). TSAb binds to TSHR to produce a biological effect similar to TSH, which is the direct cause of GD. TBAb binds to TSH and blocks the binding of TSH to receptors, inhibiting thyroid hyperplasia and thyroid hormone production. GD patients may have both stimulating and blocking antibodies coexisting, and the final outcome of their thyroid function depends on which antibody is dominant. In clinical practice, spontaneous hypothyroidism in GD patients is related to the appearance of serum TBAb. TGI binds to thyroid TSHR, and its biological effects are different from TSAb. It only stimulates thyroid cell proliferation and does not cause hyperthyroidism. Therefore, when studying the establishment of GD models, attention should be paid to the types of TRAb and their relationship with clinical manifestations and pathological changes.

　　The autoimmune GD model is suitable for the study of the pathogenesis of human GD, as well as the screening and pharmacological research of hyperthyroidism drugs.

　　Genetic background plays an important role in triggering hyperthyroidism in mice. For example, immunization of mice with adenovirus expressing TSHR resulted in elevated serum T4, TSI, and TBII levels in 55% female, 33% male BALB/c, and 25% female C57BL/6 mice. In the CBMJ (H-2k), DBA/1J (H-2q), and SJL/J (H-2s) strains, no symptoms of hyperthyroidism were observed. This has been found in both cellular immunity and nucleic acid immunity modeling.