目的在建立EAT动物模型的基础上,探索通过非病毒载体基因转染方法,以改善EAT大鼠免疫功能异常状态,并观察对大鼠脾细胞增殖及分泌Th1细胞因子的影响.方法选用♀W istar大鼠建立EAT动物模型.成模大鼠分为4组:A组(PBS+PLL),B组(pORF质粒+PLL)、C组(pOR-Fm IL10质粒+PLL)及D组(pORFm IL10质粒+MEM).使用R IA方法测定TgAb、TmAb滴度.脾脏淋巴细胞培养,采用[3H]-TdR标记方法测定cpm值并计算淋巴细胞增殖指数,ELISA方法检测细胞因子IFN-γ、TNF-α及IL-2.结果C组与A、B、D组比较,第4、6、8周TgAb、TmAb滴度降低,(P“,”Aim The autoimmune response in experimental autoimmune thyroiditis(EAT) is characterized by lymphocytes infiltration of the thyroid gland and evaluated by circulating autoantibodies specifically to thyroid antigen.Since IL-10 seems to be very effective in immunomodulation of EAT,we investigated local whether expression of IL-10 in the thyroid could alter the immune disorder condition.Methods Wistar female rats weighed 60~80 g were immunized with PTg 100 μg emulcificated with CFA subcutaneously.Enhanced immunization with the same dose of antigen emulcificated with IFA at 2 wk,3 wk and 4 wk.EAT rats were divided into four groups:group A(PBS+PLL),five rats,group B(pORF+PLL) five rats,group C(pORFm IL10+PLL) ten rats,group D(pORFmIL10+MEM) five rats.The plasmid was mixed with lipofectamine,then injected into the thyroid tissues of rats on day 18 after-plasmid immunization.The rats were sacrificed at 8wk,thyroids were mored and fixed in 10% neutral formalin solution and processed according to standard procedures.The specimens were stained with hematoxylin-eosin-safran.The level of serum TgAb and TmAb were detected at 0,2,4,6,8 wk.In vitro proliferative responses to ConA and different concentrations of PTg were measured by culturing 4*10~5 spleen cells pulsed with 18.5 KBq of thymidine for the final 12h and then harvested for liquid scintillation counting.Th1 cytokines IFN-γ,TNF-α and IL-2 were detected using ELISA.Results A dramatic lymphocytic infiltration was observed in thyroids of experimental autoimmune thyroiditis rats.The serum TgAb and TmAb of EAT rats increased at 2 wk after first immunization,but no statistical difference was found among the groups.The rats in group C had lower level of TgAb and TmAb than those of the other groups at 4,6,8 wk,(P<0.01,F=42.66,F=32.65,F=9.66;F=22.25,F=81.35,F=14.84,respectively).The proliferative response to TG was suppressed in group C than that in groups A and B(P0.05).Concentrations of IFN-γ,TNF-α and IL-2 in groups A and B were higher than those in group C in PTg-stimulated spleen cells supernatant(P<0.01,P<0.05,respectively).Conclusion Cytokines play a crucial role in the immunoregulation and pathology of experimental autoimmune thyroiditis.Systemic and local administration of IL-10 has a curative effect on EAT.To avoid the side effect of high dose and frequent use of cytokine,we devised an method of gene therapy to deliver pORFmIL10 plasmid DNA encoding IL-10 into the inflamed thyroid and exert the protective function.IL-10 can inhibit the development of lymphocytic infiltration of the thyroid.These results are paralleled by significantly decreased serum autoantibody and anti-PTg proliferation response of spleen cells in IL-10 gene transmission rats vs control groups.The data strengthens our demonstration that local IL-10 gene therapy with lipofectamine-PLL complexes can induce a fast and long-lasting expression of IL-10 on rat thyroid follicular cells and is a curative treatment of EAT in rats.The low amount of cytokine produced locally would probably minimize the side effects after high dose systemic injection.Non-viral vector gene transmission is a safe and effective method avoiding the risk of gene mutation.In conclusion,local IL-10 gene therapy using non-viral vectors is a novel and promising approach for the treatment of thyroid autoimmune disorders that may be applied in human in the future.