摘要
目的:应用RNA干扰技术(RNAi)研究针对survivin基因的siRNA,抑制survivin基因的表达并诱导食管癌细胞系EC-109细胞的凋亡.方法:构建针对survivin的siRNA表达质粒,转染至EC-109细胞,荧光显微镜判断转染效率,蛋白质印迹和半定量RT-PCR检测survivin蛋白表达及基因转录水平的变化,并在不同时间点收集转染细胞,利用流式细胞术及基因组DNA凋亡检测试剂盒,观察siRNA抑制survivin基因表达后诱导细胞凋亡的情况.结果:荧光显微镜结果表明,重组质粒转染效率达46.70%.半定量RT-PCR检测到pSIREN/S质粒在EC-109细胞内对survivin基因的转录抑制率为74.04%;蛋白质印迹结果表明,转染重组质粒pSIREN/S的EC-109细胞survivin蛋白表达量仅为正常组的41.64%,而对照质粒pSIREN/CN对survivin基因的蛋白表达及转录均没有抑制作用.经pSIREN/S转染的EC-109细胞基因组DNA出现明显的DNAladder,流式细胞仪分析结果也显示凋亡细胞为60.78%.结论:survivin特异性siRNA可明显抑制survivin基因的转录和表达,并能有效地诱导EC-109细胞的凋亡,为下一步在体内利用pSIREN/S重组质粒沉寂survivin基因,促肿瘤细胞的凋亡提供实验基础.
AIM： To inhibit the expression of endogenous survivin gene and induce the apoptosis in human esophageal cancer cell line EC-109 using small interfering RNA （siRNA） targeting survivin gene. METHODS： The recombinant plasmid pSRIEN/S was constructed and transfected into EC-109 cells. The transfection efficiency was determined by using fluorescence microscopy. The mRNA silence of survivin gene was detected by semi-quantitative RT-PCR and the expression level of survivin protein was determined by Western Blot and immunofluorescence staining. The apoptotic rate was observed by the flow cytometry and Genomic DNA Extract Kit. RESULTS： Fluorescence microscopy showed that the transfection efficiency was about 46.70%. Compared with survivin mRNA expression in the control siRNA-treated EC-109 cells, the transcription after treatment with survivin-specific siRNA was silenced by 74.04% , and the expression of survivin protein was reduced to 41.64%. The results of DNA ladder and the flow cytometry demonstrated that the apoptotic rate of EC- 109 cells was significantly increased to 60. 78%. CONCLUSION： The siRNA targeting survivin gene shows a dramatic inhibitory effect on RNA transcription and protein expression and a promoting effect on the apoptosis in EC-109 cells, which offers a reliable base for the further in vivo experiment.
出处
《第四军医大学学报》
CAS
北大核心
2007年第1期75-78,共4页
Journal of the Fourth Military Medical University
