ABSTRACT
Objective Graves' disease is the most common autoimmune thyroid disease and its prevalence and clinical manifestations are disparate between females and males. Costimulatory molecules play an essential role in regulating autoimmune responses. The objective of this study was to determine if expression of inhibitory molecules was correlated with treatment by dihydrotestosterone (DHT) in an BALB/c mouse model of experimental autoimmune Graves' disease.Methods Female BALB/c mice were immunized three times with thyroid stimulating hormone receptor A-subunit encoded by adenovirus to establish a Graves' disease model. Three different doses of DHT or a matching placebo were administered by implantation of slow-release pellets a week before the first immunization. Four weeks after the third immunization, the mice were euthanatized, and then the spleen and thymus were removed. Total thyroxine and free thyroxine levels in serum of mice were detected using a radioimmunoassay kit. Real-time polymerase chain reaction was performed to estimate the expression of costimulatory molecules in lymphocytes from the spleen and thymus. Flow cytometry was used to analyze the percentage of CD4 T cells in splenic lymphocytes. Quantitative data were compared with unpaired -tests. Correlation between two variables was analyzed using Analysis of Variance.Results Treatment with DHT can dramatically reduce total thyroxine and free thyroxine levels. Higher expression of programmed death-1 was found in the spleen of Graves' disease mice receiving 5 mg of DHT treatment (0.635±0.296 . 0.327±0.212; =2.714, =0.014), similarly, T-cell immunoglobulin domain and mucin domain 3 (TIM-3) in both the spleen (1.004±0.338 . 0.646±0.314; =2.205, =0.022) and the thymus (0.263±0.127 . 0.120±0.076; =3.221, =0.004) also increased after 5 mg of DHT treatment compared with the parallel placebo model mice. Moreover, the percentage of CD4 T cells declined in the splenic lymphocytes of Graves' disease mice treated with 5 mg of DHT (19.90%±3.985% . 24.05%±2.587%; =2.804, =0.012). A significant negative association was observed between expression of TIM-3 in the spleen and serum levels of total thyroxine (=-0.7106, =0.014) as well as free thyroxine (=-0.6542, =0.029).Conclusion This study demonstrates that DHT can ameliorate experimental autoimmune Graves' disease, which may occur by up-regulating expression of programmed death-1 and TIM-3 and inhibiting development of CD4 T cells.
ABSTRACT
Objective: To construct an adeno-associated viral vector serotype2/9 containing human thyrotropin receptor (rAAV2-9-hTSHR289-IRES-ZsGreen) so as to provide a better means for establishing an ideal animal model and the gene prevention against Graves' disease. Methods: AAV skeleton plasmid pAAV-IRES-ZsGreen and PDC315 plasmid containing hTSHR289 were digested by EcoR+BamH. The digestion products were connected. And pAAV-hTSHR289-IRES-ZsGreen vector was generated after transformation of JM109 by ligation products. The recombinant plasmid was identified by gel electrophoresis and sequencing. Three plasmids (pAAV-hTSHR289-IRES-ZsGreen, pHelper, and pAAV-2/9) were transfected into 293AAV cell line by calcium phosphate method and a large amount of rAAV2/9-hTSHR289-IRES-ZsGreen was obtained. After purification, the packaging efficiency of recombinant adeno-associated virus was observed by using fluorescence microscope, and its titer was determined by rQ-PCR. HEK293 cells were transfected with rAAV2/9-hTSHR289-IRES-ZsGreen; then the expression level of hTSHR289 was analyzed by ELISA at different time points. Results: The success of hTSHR289 gene inserted into the AAV skeleton vector was confirmed by double digestion and sequencing. After 72 h of transfection of 293AAV cells, the packaging efficiency of the virus was 92%-94% under fluorescence microscope. The titer of the recombinant adeno-associated virus was 1×1013vg/mL. ELISA assay showed that the expression level of hTSHR289 protein mediated by rAAV2/9 was significantly higher at 96 h than that at 72 h and 48 h. Conclusion: The rAAV2/9-hTSHR289-IRES-ZsGreen was successfully constructed. Furthermore, it could be transfected and expressed in cells effectively.