siRNA对人-单核巨噬细胞TLR2基因表达的抑制

摘要

To screen the siRNAs (small interference RNA sequences ) which specifically inhibit the gene expression of TLR2 in human monocyte-derived macrophage , and discuss their prospects on the treatment of HIV at the level of molecular immunology.Methods:We obtained the mRNA sequences of human TLR 2 gene from NCBI gene bank ,then designed three siRNAs by siDESIGNTM software.The siRNA targeting human housekeeping gene GAPDH was used as positive control.The fluorescent labeling missense siRNA sequences (NC-FAM ) was used as negative control.We collected fresh peripheral blood from healthy volunteers and isolated mononuclear cells from the blood samples.The human mononuclear macrophages were then purified from mononuclear cells by utilizing adherence method.Cationic liposome reagent Lipofectamine 2000 was used to mediate siRNAs into the human mononuclear macrophages.The levels of TLR2 mRNA expression of siRNA-transfected monocyte-derived macrophage were determined by q-PCR.Expression of TLR2 protein was determined by Western blot.Results: At 72 h after transfection ,we found that the expression of GAPDH mRNA and protein in positive control group decreased significantly.Also found there existed significant differences between each siRNA group (F=41.957,P<0.001).Compared with negative control ,the relative expression of TLR2 mRNA in all siRNAs groups decreased significantly (P<0.05 ) , and the inhibition rates were 46%, 43%, 43% by three miRNAs respectively.Western blot showed that the expression of TLR 2 protein in siRNAs groups decreased significantly compared with that of control (P<0.05 ).Conclusion: The designed siRNAs in this study could inhibit the expression of TLR 2 gene in human monocyte-derived macrophage ,indicating that mediation of TLR-2 expression by siRNA might be a novel strategy for HIV treatment from the per-spective of molecular immunology.%目的：筛选特异性抑制人-单核巨噬细胞TLR2基因表达的siRNA（ small interference RNA ）序列，在分子水平上初步讨论其在HIV治疗方面的前景。方法：在基因库中寻找TLR2基因的mRNA序列，针对TLR2基因设计并合成3条siRNA，阳性对照为人类看家基因GAPDH siRNA，阴性对照为6-羧基荧光素标记的siRNA（ NC-FAM）。采集健康人新鲜外周血，分离单个核细胞，再经贴壁法培养纯化为人-单核巨噬细胞。采用阳离子脂质体试剂Lipofectamine 2000介导转染培养的人-单核巨噬细胞。用q-PCR法测定干扰后单核巨噬细胞的TLR2 mRNA的表达，Western blot 法测定干扰后TLR2蛋白表达。结果：转染72 h后，阳性对照组GAPDH mRNA及蛋白的表达明显降低（ P＜0.05）。 siRNAs组TLR2 mRNA表达水平整体有显著性差异（F＝41.957，P＜0.001），与阴性对照组比较，TLR2 siRNA各组的TLR2 mRNA相对表达水平均明显降低（P＜0.05），抑制率分别为46％、43％、43％。 WB结果显示，TLR2 siRNA各组的TLR2蛋白表达量显著降低（ P＜0.05）。结论：本研究设计的siRNA能够有效抑制人-单核巨噬细胞TLR2基因的表达，从分子免疫学角度出发，表明通过阻断siRNA介导的TLR2信号传导通路能够为HIV治疗提供新思路。