小分子干扰RNA靶向抑制suvivin基因诱导U251细胞凋亡的实验研究  

Apoptosis of glioma cell line U251 induced by small interfering RNA targeting survivin

作　　者：徐如祥[1] 涂艳阳[1] 姜晓丹[1] 封江南 黄俊 

机构地区：[1]南方医科大学珠江医院神经外科/广东省神经医学研究所,广东广州510282 [2]武汉市晶赛生物工程技术有限公司,湖北武汉430074

出　　处：《南方医科大学学报》2006年第4期398-401,共4页Journal of Southern Medical University

基　　金：国家自然科学基金(30170961)~~

摘　　要：目的构建靶向survivin基因的小分子干扰RNA(siRNA)表达载体,导入人胶质瘤细胞U251中,研究siRNA靶向抑制survivin基因对U251细胞的凋亡诱导作用。方法根据siRNA设计原则,在survivin全长序列中选取设计含19个核苷酸(19nt)靶序列两条反向重复序列,间以9个核苷酸的茎环序列,两端分别加上对应的酶切位点,形成siRNA的 DNA模板并克隆到siRNA表达载体pGenesil-1中,获得靶向抑制survivin基因的siRNA表达载体pGenesil-1／survivin; 采用Metafectene转染试剂将干扰质粒导入到胶质瘤细胞U251;分别采用实时荧光定量PCR以及Western bloting从 mRNA和蛋白水平检测干扰效果;采用Annexin-V／PI双色标记的流式细胞仪法检测siRNA诱导的细胞凋亡。结果实时荧光定量PCR以及Western blotting显示:survivin基因的mRNA转录水平以及蛋白水平的表达均得到显著抑制;流式细胞仪检测结果显示:survivin基因表达被抑制后,U251细胞凋亡率明显增高。结论靶向survivin基因的重组siRNA 干扰载体pGenesi-1／survivin介导的RNAi显著靶向抑制了survivin基因在人胶质瘤细胞U251中的表达,并明显诱导了U251细胞发生凋亡。Objective To construct recombinant expression vectors of small interfering RNA （siRNA） targeting survivin and investigate apoptosis of glioma cell line U251 mediated by the survivin-targeting siRNA. Methods According to the sequence of the coding region of survivin gene, two strings of 19 nucleotides of inverted sequence flanking the loop sequence of two complementary 9-base oligonucleotides were designed and synthesized to form hairpin construct as the DNA templates for the target siRNA. The siRNA templates were cloned into siRNA expression vector pGenesil-1, and the resulted vector pGenesil-1/survivin was transfected into U251 cells using Metafectene following the standard protocols. Real-time PCR and Western blotting were performed to evaluate survivin gene silencing induced by siRNA transfection at the RNA and protein levels, respectively. Flow cytometry analysis with Annexin-V/PI double staining was used to determine the cell apoptosis. Results Real-time RT-PCR and Western blotting revealed significantly lowered survivin expression at both RNA and protein levels in transfected U251 cells, which exhibited a significantly higher apoptosis rate after transfection as shown by flow cytometry analysis. Conclusion RNA interference mediated by the siRNA expression vector pGenesi-1/survivin can significantly reduce survivin expression and induce remarkable apoptosis in U251 cells.

关 键 词：小分子干扰RNA suvivin基因 胶质瘤 U251细胞 细胞凋亡 

分 类 号：R739.41[医药卫生—肿瘤]