RNAi抑制TLR9表达影响小鼠骨髓来源树突状细胞对CpG的应答

RNA interference on the expression of TLR9 affects the response of bone marrow-derived murine dendritic cells to CpG

为了探讨特异性降低Toll样受体9(TLR9)表达后,小鼠骨髓来源树突状细胞(dendritic cell,DC)对CpG刺激的应答反应,我们合成了特异性干扰TLR9表达的小干扰RNA(small-interference RNA,siRNA),用Lipofectamine2000转入DC,利用Block-iT检测评估了转染效率;RT-PCR方法检测了干扰效果;Annexin V和PI双染检测了细胞凋亡率。进一步在CpG作用后,用流式细胞术分析了DC表面标志CD40和MHC II的表达以及DC吞噬FITC-dextran(右旋糖酐)的能力。结果显示,siRNA转染效率较高,TLR9表达被显著下调。干扰TLR9后DC在CpG作用下,表面标志CD40和MHCII表达未被上调,但吞噬功能维持较强的水平,DC表现出未成熟状态。这些结果提示,我们建立的RNA干扰(RNA in-terference,RNAi)方法能够有效阻断TLR9的表达,为进一步研究CpG信号刺激对DC成熟和功能变化的影响提供了一种有效工具。

In the present study, the inhibitory effect of RNA interference on the expression of Toll-like receptor （TLR9） in the bone marrow derived murine dendritic cells （BMDC） was investigated and the phenotype and endocytosis of these dendritic cells （DCs） after CpG stimulation were detected. The small interference RNA （siRNA） with specific interfering effect to the expression of TLR9 was synthesized and it was transferred to DCs by lipifectamine 2000. The efficacy of transfection was eval uated by use of Block-iT. meanwhile, mRNA expression of TLR9 was analyzed by RT-PCR and the cell viability of the tras-fected DCs was analyzed by double staining with Annexin V and propidium iodine. After transfection, DCs were treated with CpG ODN for 24 hours and then the expression of the surface marker CD40 and MHC II molecules on DCs and the ability of DCs to gulf FITC-dextran were analyzed by flow cytometry. It was demonstrated that the efficacy of transfection with siRNA was proved to be rather high with marked down-regulation of the TLR9 expression without affecting to the cellular viability. The stimulation with CpG ODN increased the expressions of MHC II and CD40 molecules in the cytomembrane of DCs and reduced their endocytosis. Also, the RNA interference（RNAi） could inhibit those effects induced by CpG. It is evident that the RNAi method established in our laboratory can block effectively the expression of TLR9, thus providing a useful tool for the study on the influence of CpG signal stimulation in the maturation and functional changes of DCs.