siRNA对人-单核巨噬细胞TLR2基因表达的抑制

发布时间：2018-12-16 19:42

【摘要】：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(P0.05)。siRNAs组TLR2 mRNA表达水平整体有显著性差异(F=41.957,P0.001),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(P0.05),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(P0.05)。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。
[Abstract]:Aim: to screen the siRNA (small interference RNA) sequence that specifically inhibits the expression of TLR2 gene in human mononuclear macrophages, and to discuss its prospect in the treatment of HIV at molecular level. Methods: the mRNA sequence of TLR2 gene was searched in the gene bank. Three siRNA, positive controls were designed and synthesized for TLR2 gene. The siRNA (NC-FAM) labeled with 6-carboxyl fluorescein was used as the GAPDH siRNA, negative control of human housekeeping gene. Mononuclear cells were isolated from fresh peripheral blood of healthy people and purified from human mononuclear macrophages by adherent method. Human mononuclear macrophages were transfected with cationic liposome reagent Lipofectamine 2000. The expression of TLR2 mRNA in mononuclear macrophages after interference was determined by q-PCR method. The expression of TLR2 protein was detected by, Western blot method. Results: after 72 hours of transfection, the expression of GAPDH mRNA and protein in the positive control group was significantly lower than that in the negative control group (P0.05). There was a significant difference in the TLR2 mRNA expression level between the). SiRNAs group and the negative control group. The relative expression level of TLR2 mRNA in TLR2 siRNA group was significantly decreased (P0.05), and the inhibition rate was 46% and 43kW respectively. The results showed that the expression of TLR2 protein in TLR2 siRNA group was significantly decreased (P0.05). Conclusion: the designed siRNA can effectively inhibit the expression of TLR2 gene in human mononuclear macrophages. From the point of view of molecular immunology, it is suggested that blocking the TLR2 signal transduction pathway mediated by siRNA can provide a new idea for HIV therapy.
【作者单位】：广西艾滋病防治研究重点实验室广西医科大学公共卫生学院;广西医科大学第一附属医院老年消化内科;
【基金】：国家自然科学基金资助项目(31360033) 广西教育厅高校科研重点资助项目(2013ZD012)资助
【分类号】：R392

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