Survivin shRNA重组慢病毒构建及对A549细胞增殖的影响  

Effect of shRNA Survivin-mediated Inhibition of Proliferation in A549 Cells

作　　者：武丽红[1] 王金河[1] 王茜[1] 贾懂懂[1] 梁建琴[1] 

机构地区：[1]中国人民解放军第309医院结核病研究所,北京100091

出　　处：《中国肺癌杂志》2011年第12期903-907,共5页Chinese Journal of Lung Cancer

摘　　要：背景与目的 Survivin是凋亡抑制蛋白(inhibitor ofapoptosis protein,IAP)家族的成员,在多种肿瘤组织中高度表达而在终末分化细胞中极少表达,因此可以作为癌症治疗的理想靶点。本研究旨在通过构建Survivin基因shRNA的慢病毒质粒并干扰肺癌细胞A549中Survivin的表达,分析其对细胞增殖的影响。方法设计Survivin干扰靶序列,构建重组质粒;将pLL3.7-Survivin转染293T细胞后利用Hela细胞检测病毒的滴度并感染A549细胞,应用RTPCR和Westernblot检测干扰效果;MTT与流式细胞术分析其对细胞增殖的影响。结果本研究成功构建了重组质粒;重组质粒可抑制A549细胞中Survivin基因的表达;细胞受阻于G_2/M期。结论本研究构建的重组质粒可抑制Survivin基因的表达并影响细胞的增殖,其为研究RNAi介导的肺癌基因治疗打下基础。Background and objective Survivin is a member of the inhibitor of apoptosis family of proteins.The Survivin protein is highly expressed in most human tumors,but it is completely absent in terminally differentiated cells.Consequently, Survivin is an ideal target for cancer therapy because cancer cells are targeted and normal cells are left alone.The aim of this study is to construct a lentivirus-shRNA vector,and to disrupt the expression of Survivin in A549 cells.The effect of shRNA Survivin on A549 cells was analyzed.Methods Target DNA sequences of Survivin shRNA were designed to obtain recombinant plasmids.After the plasmids were transfected into 293T cells,the virus was collected.Hela cells were used to detect the virus titer.Survivin mRNA and protein expression in the infected AS49 cells were detected via reverse transcription polymerase chain reaction and Western blot analysis.The proliferation of A549 cells were detected via 3-（4,5-dimethylthiazolyl）- 2,5-diphenyltetrazolium bromide and flow cytometry assays.Results The recombinants were successfully constructed,and Survivin expression was inhibited.The cells were blocked at the G_2/M phase.Conclusion Recombinant lentivirus with shRNA targeting Survivin was successfully constructed.The lentivirus can down-regulate Survivin expression in A549 cells as well as inhibit proliferation,and is hence a potential gene therapy for lung cancer.

关 键 词：慢病毒 SURVIVIN 短发夹RNA 增殖 

分 类 号：R734.2[医药卫生—肿瘤]