摘要
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.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2011年第8期681-685,695,共6页
Chinese Journal of Immunology
基金
国家科技部"重大新药创制"科技重大专项(2009ZX09103-705)资助
关键词
CD86
SIRNA
免疫耐受
共刺激分子
CD86
small interfering RNA
Immune torlerance
Costimulatory molecules