靶向 survivin 基因的 shRNA 慢病毒载体的构建及其对膀胱癌 EJ 细胞凋亡的影响  

Construction and identification of survivin shRNA lentivirus vector and its effects on apoptosis of bladder cancer EJ cells

作　　者：陶燕[1] 付生军[1] 洪梅[1] 王志平[1] 马宝良[1] 卢建中[1] 

机构地区：[1]兰州大学第二医院泌尿外科研究所,甘肃省泌尿系统疾病研究重点实验室,甘肃省泌尿系临床医学中心,甘肃兰州730030

出　　处：《暨南大学学报（自然科学与医学版）》2015年第3期239-245,共7页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：甘肃省科技计划项目(1308RJYA056);甘肃省泌尿系疾病临床医学中心开放课题基金项目(mnlczxkf-28);兰州大学中央高校基本科研业务费项目(lzujbky-2014-165)

摘　　要：目的:构建survivin基因特异性shRNA慢病毒干扰载体,转染膀胱癌EJ细胞,研究survivin基因在膀胱癌细胞株中的表达抑制情况,观察survivin shRNA慢病毒载体对EJ细胞凋亡的影响.方法:以survivin基因为靶标设计shRNA干扰序列,克隆至p SIH1-H1-cop GFP慢病毒载体.干扰载体鉴定正确后转染膀胱癌细胞株EJ细胞,荧光显微镜下观察GFP表达情况,实时荧光定量PCR检测survivin基因mRNA含量变化,Western blotting法检测survivin蛋白表达,Annexin V-FITC/PI双染法检测EJ细胞凋亡情况.结果:PCR扩增鉴定、DNA测序证实survivin慢病毒干扰载体构建成功;EJ细胞经干扰载体处理后,EJ细胞survivin基因mRNA水平下调了73.33%,蛋白表达受到显著抑制,EJ细胞凋亡率达到24.39%.结论:成功构建了靶向survivin基因的shRNA重组慢病毒干扰载体,可以显著降低转染细胞survivin基因的表达水平,并有效提高膀胱癌细胞凋亡率.Aim：To construct an effective survivin gene shRNA lentiviral vector,and evaluate its effects on apoptosis of bladder cancer cell line EJ.Methods：The shRNA template of survivin was cloned into pSIH1-H1-copGFP shRNA vectors.The recombinant plasmid was identificated by agarose gel elec-trophoresis and sequencing analysis;then it was transfected into the EJ cells.The mRNA and protein lev-els were measured using real-time quantitative PCR and Western blotting assay respectively,and the ap-optosis of EJ cells was measured by Annexin V-FITC /PI double staining assay.Results：The survivin gene shRNA lentivirus vector plasmid was successfully constructed.Agarose gel electrophoresis and se-quencing analysis confirmed the correct of plasmid construction.After shRNA plasmid transfection,the mRNA levels of survivin was significantly suppressed（suppression rate 73.33%,P 〈0.05）,and the pro- 〈br〉 tein expression was decreased significantly.The apoptosis rate in the shRNA treated group was dramatic-ally increased when compared to the control group.Conclusion：The survivin shRNA lentiviral vector sig-nificantly suppressed survivin expression and induced apoptosis of bladder cancer EJ cells.

关 键 词：载体构建 SURVIVIN 短发卡状RNA(shRNA) 膀胱癌 细胞凋亡 

分 类 号：R737.14[医药卫生—肿瘤]