| Objective: To investigate the methods for developing animal model of TAO, and study the mechanism of TAO.
Methods：Constructed competence cells of bacterium coli using CaCl2, and transfected competence cells of bacterium coli with plasmid pcDNA3.1/hTSHR and acquired positive clones. 43 BALB/c mice were randomly divided into experimental group, empty plasma control group and normal control group. Injected the BALB/c mice with plasmid pcDNA3.1/hTSHR at the different injection location at 0 week(wk), 3wks, 6wks and 9wks in experimental group, and injected the mice with plasmid pcDNA3.1 at different time point in empty plasma group, and observed their weight at every time point. At the end of 18 weeks, all mice were sacrificed, their sera were used for measuring total T4 and TRAb by ELISA. Their thyroid glands and orbital tissues were removed for histopathological examination, and Th1/Th2 response were dectected in orbital tissues by immuno- histochemical method.
Results：All mice weight increased. Only the weight of the groups at anterior tibialis muscles injection and at both the right orbit and anterior tibialis muscle injection increased slowly. The serum total T4 and TRAb level of all immunized mice were within the normal reference value. Almost all the serum total T4 and TRAb level in the experimental group decreased at different degrees compared with controls, however, increased compared with empty plasmid group. Thyroiditis appeared in most of the experimental mice. Some mice presented lymphocytic infiltration with the appearance of lymphocytic nests. The corresponding orbital pathologic changes appeared in 41.2% mice immunized with plasmid pcDNA3.1/hTSHR, which included: edema of orbital tissues, accumulation of orbital fat, infiltration of immunocytes, et al. Hypertrophy of the cross-section of the extraocular muscular fibers, degeneration, breakage of muscular fibers with unclear circumferences, endomysium and perimysium were infiltrated with dark prunosus mast cells at different degrees. Orbital fat and connective tissues were infiltrated with inflammatory cells. In the experimental group, the extraocular muscles showed that derangement, breakage of the muscular fibers. Some Z lines weren’t clear and infiltrated with plasma cells under transmission electron microscope. At 4wk, almost orbital lymphocytic infiltrations were Th1 immune response, while those were Th2 response at 8 wk.
Conclusion：To develop TAO animal model with TSHR gene immunization is an effective method. The orbital histological characteristics of TAO model animal are similar to that of human TAO. In early stage, almost orbital lymphocytic infiltrations were Th1 immune response, while those were Th2 response in later stage.
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