IL-10基因转染对EAT大鼠脾细胞增殖及Th1细胞因子分泌的影响

The effect of IL-10 gene transmission into thyroid tissues on the PTg-stimulated splenocytes proliferation and Th1 cytokines production in rats

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<0.01,F=42.66,F=32.65,F=9.66;F=22.25,F=81.35,F=14.84)。此外,淋巴细胞增殖试验结果显示,C组PTg刺激的淋巴细胞增殖指数低于A,B组,(P<0.05)。PTg刺激的脾细胞培养上清中细胞因子IFN-γ、TNF-α及IL-2检测结果,A、B组高于C组(P<0.01,P<0.05)。结论通过大鼠甲状腺组织内局部注射pORFm IL10质粒使甲状腺滤泡上皮细胞表达IL-10,能够降低自身抗体水平和针对抗原反应的T淋巴细胞增殖反应及PTg刺激的脾细胞产生的Th1细胞因子。多聚赖氨酸可以延长目的基因表达时间,更有效地改善EAT大鼠的免疫功能状态。

Aim The autoimmune response in experimental autoimmune thyroiditis （EAT） is characterized by lymphocytes infiltration of the thyroid gland and evaluated by circulating autoantihodies 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 100μg 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 （pORFm IL10 ＋ 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 [^3H ] thymidine for the final 12h and then harvested for liquid scintillation counting. Thl 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 （P〈0.05）. There was no different response to ConA a- mong the groups （P 〉 0. 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 devel- opment 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 cytoklne 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.