特异性hTERT启动子在肿瘤细胞中启动效率研究  

Activity of human telomerase catalytic subunit promoter in tumor cell lines

作　　者：李玮[1] 谭建[1] 吴蓓[1] 李宁[1] 

机构地区：[1]天津医科大学总医院核医学科,天津300052

出　　处：《生物医学工程与临床》2010年第3期252-256,共5页Biomedical Engineering and Clinical Medicine

基　　金：天津市应用基础及前沿技术研究计划资助(08JC2DJC23900)

摘　　要：目的证明人端粒酶亚单位(hTERT)启动子可以引导目的基因在肿瘤细胞中表达。方法先使用RT-PCR检测各细胞系中hTERT mRNA的表达,再使用PCR方法扩增hTERT启动子基因的不同长度片段,并构建重组质粒,使用BglⅡ和HindⅢ双酶切鉴定重组质粒并测序。最后使用Dual-Glo Luciferase Assay System激发荧光检测各细胞系中重组质粒中hTERT启动子的启动效率。结果 pGL3-204、pGL3-378、pGL3-1375重组质粒中的hTERT启动子序列与预期一致,质粒构建成功。hTERT启动子引导荧光基因的重组质粒在H460中启动效率最强可以达到SV40启动子的80%,而在FRO、U251中启动效率可以达到SV40启动子的30%左右。hTERT全长启动子序列的启动效率在ARO细胞系中比pGL3-204、pGL3-378中的启动子片段启动效率强,而在FRO、H460和U251细胞系中pGL3-204中的启动子片段启动效率最强。结论 hTERT启动子引导荧光基因的重组质粒,可以实现只在肿瘤细胞系中表达,但在正常细胞中不表达的效果。Objective To prove the human telomerase catalytic subunit（hTERT） promoter guided target gene expression in tumor cells.Methods The reverse transcription polymerase chain reaction（RT-PCR） was performed to detect the expression of hTERT mRNA in tumor cells and human normal cells.Different lengths of hTERT promoter were amplified from genome cDNA by polymerase chain reaction（PCR）, and were inserted into luciferase reporter vectors（pGL3） to construct recombinant plasmid, then identified recombinant plasmid by BglⅡand HindⅢ and transcriptional activities of hTERT promoter in cell lines by measuring the luciferase activities.Results The hTERT promoter in pGL3-204,pGL3-378,pGL3-1375 recombinant plasmid was detached as the expected results and the final formation could be achieved.The activity of recombinant plasmid had strong expression of luc＋ in ARO cell lines, and reached to as much as 60 % of SV40.In FRO,U251 cell lines the figure was 20 % of SV40 promoter activity.Conclusion The hTERT promoter guided target gene has different transcriptional activities in various tumor cells, but no transcriptional activity in normal cells.

关 键 词：人端粒酶亚单位启动子 肿瘤 重组质粒 

分 类 号：R73-3[医药卫生—肿瘤]