PLAGL2基因siRNA表达质粒的构建及体外干扰作用

Construction of PLAGL2 siRNA Expression DNA Plasmid and the Interfering Effect on Gene Expression in H441 Cells

目的构建PLAGL2 siRNA表达载体质粒并探讨其体外干扰作用。方法构建的PLAGL2 siRNA表达载体质粒经脂质体介导转染到小鼠肺Ⅱ型上皮细胞株H441细胞中并获得稳定细胞株,采用实时荧光PCR及Westernblot技术分别检测转染组和野生型H441细胞中PLAGL2、SP-C、SP-B mRNA和PLAGL2蛋白表达水平的变化。结果实时荧光PCR检测结果显示:与野生型H441细胞相比较,PLAGL2 siRNA表达载体质粒转染的H441细胞中PLA-GL2 mRNA水平明显降低(P<0.05),SP-C mRNA水平明显增高(P<0.05),而SP-B mRNA水平没有明显差异(P>0.05);Western blot检测结果显示:与野生型H441细胞相比较,PLAGL2 siRNA表达载体质粒转染的H441细胞中PLAGL2蛋白表达水平明显降低(P<0.05)。结论成功构建了PLAGL2 siRNA表达载体,将其转染至H441细胞中可有效抑制PLAGL2基因的表达,同时促进SP-C基因的表达。

Objective To construct human PLAGL2 siRNA expression DNA plasmid and study the interfering effect on gene expression in H441 cells.Methods H441 cells were transfected with PLAGL2 siRNA expression DNA plasmid with Fugene6.Real time PCR and Western blot were employed to detect the interfering effect on the expression of PLAGL2,SP-C,SP-B mRNA and protein in wildtype and PLAGL2 siRNA expression DNA plasmid transfected H441 cells respectively.Results Real time PCR revealed that,as compared with wildtype H441 cells,the expression level of PLAGL2 mRNA in PLAGL2 siRNA plasmid transfected H441 cells was significantly reduced（P〈0.05）,but that of SP-C mRNA in PLAGL2 siRNA plasmid transfected H441 cells was significantly increased（P〈0.05）.Western blot showed that,as compared with wildtype H441 cells,the expression level of PLAGL2 protein in PLAGL2 siRNA DNA plasmid transfected H441 cells was significantly reduced（P〈0.05）.Conclusion Human PLAGL2 siRNA expression DNA plasmid was constructed successfully,and its transfection into the H441 cells could effectively inhibit the PLAGL2 expression,and simultaneously promote the expression of SP-C.