靶向survivin的siRNA表达载体的构建和表达及对HeLa细胞的影响  

RNA interference-mediated inhibition of survivin expression in Hela cell line by siRNA expression vector targeting survivin gene

作　　者：蒋鸿超[1] 孙强明[1] 赵丹[2] 孙茂盛[1] 吕琳[2] 李鸿钧[1] 

机构地区：[1]中国医学科学院/中国协和医科大学医学生物学研究所分子生物室,云南昆明650118 [2]昆明医学院研究生部,云南昆明650031

出　　处：《南方医科大学学报》2006年第12期1806-1811,共6页Journal of Southern Medical University

摘　　要：目的应用RNA干扰技术(RNAi)针对凋亡抑制因子survivin存活素的siRNA抑制Hela细胞株内源survivin基因的表达以及可促进Hela细胞凋亡。方法构建重组质粒pshRNA-survivin-1和pshRNA-survivin-2并分别转染Hela细胞株,通过免疫荧光和半定量RT-PCR检测survivin蛋白表达及mRNA转录水平的变化,并通过流式细胞仪使用PI-AnnexinV双染法检测Hela细胞株的凋亡。结果构建的2种重组质粒pshRNA-survivin-1和pshRNA-survivin-2均能明显抑制survivin基因的表达;应用免疫荧光检测survivin基因的表达,转染重组质粒pshRNA-survivin-1和pshRNA-survivin-2的实验组survivin荧光强度明显低于转染空载体pGE-1和pshRNA阴性非特异对照质粒;通过半定量RT-PCR检测到survivin基因mRNA转录明显减少;通过PI-AnnexinV双染法检测Hela细胞的凋亡分别达(36.02±2.12)%(P<0.01)和(35.29±2.02)%(P<0.01)。结论重组质粒pshRNA-survivin-1和pshRNA-survivin-2均可明显抑制Hela细胞内源survivin的表达和mRNA的转录均可并明显促进Hela细胞的凋亡,为survivin介导的肿瘤基因沉寂疗法提供良好的实验基础。Objective To prepare small interfering RNA （siRNA） targeting survivin for inhibition of endogenous survivin gene expression in Hela cell line and evaluate its effect on promoting Hela cell apoptosis. Methods The recombinant plasmid pshRNA-survivin-1 and pshRNA-survivin-2 were constructed and transfected into Hela cells, in which the expression level of survivin was determined by immunofluorescence staining and survivin gene transcription detected by semi-quantitative RT-PCR. Results Introduction of the plasmids pshRNA-survivin-1 and pshRNA-survivin-2 into Hela cells resulted in efficient and specific inhibition of survivin expression as demonstrated by immunofluorescence staining. Semi-quantitative RT-PCR showed that mRNA transcription of survivin gene was reduced. In contrast, the control plasmid did not exhibit any inhibitory effect on the protein expression and mRNA transcription of survivin gene. PI-Annexin V staining indicated an apoptosis rate of the transfected Hela cells of （36.02±2.12）% （P〈0.01） and （35.29±2.02）% （P〈0.01）, respectively. Conclusion The prepared siRNA targeting survivin gene is capable of inducing marked inhibitions of survivin protein expression and RNA transcription and significant enhancement of apoptosis in Hela cells, which shed light on a new strategy in gene silence therapy targeting survivin.

关 键 词：SIRNA HELA细胞株 SURVIVIN基因 质粒 表达 

分 类 号：Q78[生物学—分子生物学]