Survivin启动子的克隆及其在膀胱癌细胞BIU87中的活性鉴定  

Molecular cloning of Survivin gene promoter and identification of its specific activities in bladder cancer cell line BIU87

作　　者：严泽军[1] 程跃[1] 蒋军辉[1] 胡嘉盛[1] 施小东[1] 黄坚成[1] 

机构地区：[1]宁波市第一医院,宁波315010

出　　处：《现代实用医学》2011年第11期1214-1216,1219,共4页Modern Practical Medicine

基　　金：浙江省自然科学基金项目(Y2090805)

摘　　要：目的克隆Survivin启动子的有效片段,并检测其在人膀胱癌细胞株BIU87和人正常膀胱上皮细胞SV-HUC-1中的转录活性。方法用PCR扩增Survivin基因的启动子片段,克隆入荧光素酶报告质粒pGL3-Basic,构建pGL3BSurvivin重组质粒,用脂质体法瞬时转染BIU87及SV-HUC-1中,检测Survivin启动子在细胞中的转录活性。同时构建含CMV启动子的pGL3BCMV重组质粒作为阳性对照。48h后收集转染细胞与荧光素酶底物反应,检测荧光素酶活性。结果成功克隆440 bp的Survivin基因启动子,并构建了携带有Survivin基因启动子的pGL3Basic真核表达载体,转染BIU87细胞后的荧光素酶活性为2 286.98±440.21,而SV-HUC-1荧光素酶活性为12.32±1.16,两者差异有统计学意义(<0.01)。结论本实验成功克隆的Survivin启动子在BIU87细胞中表现出较高的肿瘤特异性活性,其有可能作为调控元件用于膀胱癌的靶向性基因治疗。Objective To clone DNA sequence of Survivin promoter,and detect its specific transcriptional activities in bladder cancer cell line BIU87 and normal bladder cell SV-HUC-1.Methods The fragment of Survivin promoter was acquired by PCR amplification and inserted into lucirerase reporter vectors（pGL3-Basic） to reconstruct a recombinant plasmid named pGL3BSurvivin.Then the reconstructed plasmid was transiently transfected into bladder cancer cell line BIU87 and normal bladder cell SV-HUC-1.The transcriptional activities of Survivin promoter in various cells were determined by measuring the luciferase activities after 48 hours of transfection.A recombinant plasmid pGL3BCMV,containing the CMV promoter controlled luciferase gene,was used as positive control.Results A 440 bp gene fragment was amplified by PCR method from BIU87 cell genomic DNA and pGL3BSurvivin vector was constructed successfully.The activity of luciferase reporter gene was（2286.98±440.21） in BIU87 cell and（12.32±0.16） in SV-HUC-1 cell 48 hours after transfection of pGL3B Survivin vector.The differences was significant（P=0.00043）.Conclusions The high specific activities of constructed Survivin promoter eukaryotic expression vector might be a potential therapeutic reagent in the gene therapy of bladder cancer.

关 键 词：SURVIVIN 启动子 膀胱肿瘤 基因治疗 

分 类 号：R737.14[医药卫生—肿瘤]