论文部分内容阅读
目的:将Mcl-1shRNA转染到Raw264.7细胞内,针对shRNA对小鼠Raw264.7巨噬细胞系中Mcl-1表达的影响,筛选出沉默Mcl-1基因效果最明显的特异性shRNA真核表达质粒。方法:将特异性shRNA经脂质体介导转染小鼠巨噬细胞系Raw264.7;半定量RT-PCR和Western blot分别检测转染24、48 h后Mcl-1 mRNA水平变化和Mcl-1蛋白表达情况,分析对应不同位点的三对特异性shRNA片段对Mcl-1的沉默效果。结果:特异性shRNA片段在24、48 h均能有效降低Mcl-1 mRNA和蛋白水平,沉默效率高于正常组、脂质体组和阴性对照组,差异具有统计学意义(P<0.05);对应不同位点的三对shRNA真核表达质粒,其中Mcl-1 shRNA3对Mcl-1 mRNA和蛋白的抑制作用均最强。结论:RNA干扰技术可有效下调小鼠Raw264.7巨噬细胞系中Mcl-1 mRNA水平,明显下调Mcl-1蛋白表达。成功筛选出了沉默Mcl-1基因效果最明显的特异性shRNA真核表达质粒。
Objective: The Mcl-1shRNA was transfected into Raw264.7 cells, and shRNA targeting Mcl-1 expression in Raw264.7 macrophage cell line was screened out, and the specific shRNA with the most obvious effect of silencing Mcl-1 gene was screened out Nuclear expression plasmid. Methods: The specific shRNA was transfected into mouse macrophage cell line Raw264.7 by lipofectamine. Mcl-1 mRNA and Mcl-1 mRNA were detected by semi-quantitative RT-PCR and Western blot respectively. 1 protein expression analysis of the corresponding three different pairs of specific shRNA fragments of Mcl-1 silencing effect. Results: The specific shRNA fragments could effectively reduce the mRNA and protein levels of Mcl-1 at 24 h and 48 h, and the silencing efficiency was higher than that of the normal group, liposome group and negative control group (P <0.05). Three pairs of shRNA eukaryotic expression plasmids corresponding to different sites, of which Mcl-1 shRNA3 Mcl-1 mRNA and protein inhibition were the strongest. Conclusion: RNA interference can effectively down-regulate Mcl-1 mRNA and down-regulate the expression of Mcl-1 in Raw264.7 macrophage cell line. The specific shRNA eukaryotic expression plasmid with the most obvious effect of silencing Mcl-1 gene was successfully screened out.