siRNA沉默抗原递呈细胞表面CD86的表达对T淋巴细胞激活的影响

Effect of silencing CD86 expression on antigen presenting cells by siRNA on activation of T lymphocytes

目的:通过小干扰RNA(small interfering RNA,siRNA)沉默抗原递呈细胞(Antigen presenting cells,APC)表面共刺激分子CD86的表达,分析其对T细胞增殖和白介素10及IFNγ-分泌的影响,为移植排异和自身免疫性疾病的治疗提供新的思路和方法。方法:设计并通过化学方法合成三对针对人CD86 mRNA的siRNA片段(siRNA-1、2、3),脂质体法转染人B淋巴瘤Raji细胞;转染24、48、72小时后,流式细胞术(Flow cytometry,FCM)检测Raji细胞表面CD86蛋白水平的表达;荧光定量PCR方法检测CD86mRNA水平的表达;分别采用siRNA抑制Raji细胞表面CD86分子的表达、抗人CD86单克隆抗体封闭CD86分子以及二者的联合来阻断CD86-CD28信号通路、MTT和ELISA方法分别检测CD86信号通路被抑制后对T细胞增殖和细胞因子IL-10及IFNγ-分泌的影响。结果:FCM结果显示,Raji细胞表面天然表达CD86的阳性率约为98.0%;转染siRNA后24小时时,仅siRNA-2组表现较为明显的抑制效应,此时细胞表面CD86的阳性表达率约为61.7%,抑制率为37.0%;转染后48小时,control和siRNA-1组Raji细胞表面CD86的表达仍没有变化,而siRNA-2组CD86的表达下降至39.6%,抑制率为59.6%,siRNA-3组CD86的表达下降至72.6%,抑制率为25.9%;siRNA转染后72小时,各组Raji细胞表面CD86的表达均有不同程度的变化,分别为:control组,90.4%;siRNA-1组,83.1%;siRNA-2组,15.2%和siRNA-3组,46.2%;各组的抑制率依次为7.6%,15.2%,84.5%及52.8%。荧光定量PCR结果显示,转染后72小时,仅siRNA-2和siRNA-3组CD86 mRNA水平的表达与Raji细胞相比有显著差异,抑制率分别为78.7%和45.9%。CD86的表达被siRNA-2抑制后,可抑制Raji细胞对T细胞的活化、增殖和IL-10及IFNγ-的分泌;抗人CD86单克隆抗体同样可以抑制Raji细胞对T细胞的激活;并且siRNA-2和抗人CD86单克隆抗体对T细胞的活化抑制具有协同效应。结论:通过siRNA沉默抗原递呈细胞表面共刺激分子CD86的表达,从而阻断CD86/CD28共刺激信号通路,可有效抑制T淋巴细胞的活化、增殖及细胞因子IL-10、IFNγ-的分泌,由此削弱了T细胞应答来诱导免疫耐受。

Objective：To investigate the effect of silencing CD86 expression on antigen presenting cells（APC） by siRNA on proliferation and interleukine（IL）-10,IFN-γ production of T lymphocytes,and provide new methods for transplant rejection and autoimmune disease.Methods：Three siRNA sequences used for targeted silencing of human CD86 were designed and chemically synthesized,then transferred into the Raji cells by LipofectamineTM 2000;24 h,48 h and 72 h after transfection,expression of CD86 on Raji cells was assessed by flow cytometry（FCM）,and the mRNA expression was analyzed by fluorescent quantitation PCR;Inhibition the expression of CD86 on Raji cells by siRNA,blocking CD86 molecule by anti-CD86 monoclonal antibody and combination of the two methods that result in blocking CD86-CD28 signal pathway,the proliferation and IL-10,IFN-γ production of T lymphocytes were evaluated by MTT assay and ELISA.Results：The results of FCM showed：natural CD86 expression on Raji cells was 98.0%;24 h after siRNA transfection,only siRNA-2 showed apparent suppression effect,CD86 expression was 61.7%,inhibition ratio was 37.0%;48 h after transfection,CD86 expression of control and siRNA-1 groups was unchanged,that of siRNA-2 and siRNA-3 groups was 39.6% and 72.6% respectively,inhibition ratio was 59.6% and 25.9% respectively;72 h after transfection,CD86 expression of each group was：control,90.4%;siRNA-1,83.1%;siRNA-2,15.2%;siRNA-3,46.2%;inhibition ratio was 7.6%,15.2%,84.5% and 52.8% respectively.The results of fluorescent quantitation PCR showed：72 h after transfection,relative expression of CD86mRNA of siRNA-2 and siRNA-3 groups had significant deviation with normal group,inhibition ratio was 78.7% and 45.9% respectively.Suppression CD86 expression on Raji cells by siRNA-2 can effectively inhibit the activation,proliferation and IL-10,IFN-γ production of T lymphocytes as well as anti-human CD86 monoclonal antibody,and they had synergistic effect on T lymphocyte suppression.Conclusion：Silencing CD86 expression on APC by siRNA can block CD86/CD28 costimulatory pathway,inhibit the activation,proliferation and IL-10,IFN-γ production of T lymphocytes,impair the response of T lymphocytes and induce immune tolerance.