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Graves opthalmopathy (GO) is an autoimmune disease, and its pathogenesis is not clear.Recent reports found that the imbalance of Th1/Th2 type immune response may be a main cause of GO.
This study was to establish an animal model of GO by immuning female BALB/c mice with recombinant plasmid of human thyroid-stimulating hormone receptor (hTSHR) A-subunit encapsulated with liposome induces.
Thirty-two female BALB/c mice aged 6-8 weeks were randomized to recombinant plasmid group, blank control group, empty plasmid group and liposome group.Recombinant plasmid pcDNA3.1+ /hTSHR289 containing 30 μg liposome was firstly injected into pretibial muscle and then into peritoneal cavity to construct the GO models at 0, 3, 6 weeks respectively in the recombinant plasmid group, and pcDNA3.1+ and liposome was used in the same way in the empty plasmid group and liposome group, respectively.No any intervene was performed in the blank control group.Heart blood of the mice was collected before injection and 4 days and 17 weeks after injection for the detection of serum interleukin (IL)-2, IL-4 and IL-2/IL4.The mice in the recombinant plasmid group were sacrificed at the end of 17 weeks of initial injection, and thyroids and orbital tissues were extracted for the regular histopathological examination.The expressions of CD20 for B cell specific markers (B) and CD3ε for T cell specific markers (T) were detected by immunochemistry and B∶T>1.0 was Th2 response, and B∶T<1.0 was Th1 response.
Serum IL-2 levels in the recombinant plasmid group were significantly lower than those after injection of the 2nd time and 3rd time in the blank control group, empty plasmid group and liposome group (all at P<0.05), but no intergroup difference was found in serum IL-4 level (P>0.05). The serum IL-2 levels after injection of the 2nd and 3rd time were lower than those before injection and after injection of the 1st time in the recombinant plasmid group (before injection: P=0.009, 0.019; after injection of the 1st time: P=0.002, 0.004), and the serum IL-2 level raised at the sacrifice of the mice, which was significantly higher than that after injection of the 3rd time( [44.31±1.77]pg/ml vs. [42.43±2.29]pg/ml) (P=0.031). Serum IL-4 levels and IL4/IL2 values after injection of the 2nd time and 3rd time were significantly higher than those before injection and after injetion of the 1st time in the recombinant plasmid group, blank control group, empty plasmid group and liposome group, and in the sacrifice of the mice, the serum IL-4 level was higher than that after injection of the 3rd time in the recombinant plasmid group (all at P<0.05). The B∶T values in 4 of 12 orbital tissue specimens of 7 mice that showed the inflammatory cell infiltration by regular histopathology were >1.0.
Immunization syngeneic BALB/c mice with liposome encapsulated thyrotropin receptor extracellular domain recombinant plasmid is an effective way to establish a GO animal model.The Th1 type response appears in initial 17 weeks following the first immunization, but the immune balance shifts toward a Th2 type after the second immunization.