凋亡抑素启动子介导肿瘤特异性TRAIL在宫颈癌Hela细胞中的表达

目的:探讨凋亡抑素(survivin,SVV)启动子调控的TRAIL肿瘤细胞内特异表达在宫颈癌治疗中的应用意义。方法:提取人外周血淋巴细胞总RNA,采用RT-PCR方法,用带有IL-2信号肽基因序列的引物扩增TRAIL基因,克隆入真核表达载体pGL-Basic/surp中SVV启动子的下游,构建肿瘤特异性分泌表达TRAIL基因的真核表达载体pGL-Basic/surp/TRAIL。转染宫颈癌Hela细胞及正常人肝细胞(7702),RT-PCR鉴定转染细胞中的TRAIL表达。结果:RT-PCR扩增得到了613bp的cDNA片断,pGL-Basic/surp/TRAIL经酶切及测序鉴定与GenBank中报道的IL-2信号肽和TRAIL凋亡诱导功能区cDNA序列完全一致;转染细胞RT-PCR鉴定结果显示,Hela细胞中可扩增出600bp基因片段,而7702细胞中未见该特异性扩增条带。结论:重组真核表达载体pGL-Basic/surp/TRAIL能够高效、特异性地表达于Hela细胞中,其成功构建为特异性肿瘤基因治疗提供了可行的理想工具。

Expression of TRAIL mediated by survivin promoter in cervical cancer Hela cell

Objective: To explore the potential use of TRAIL expression vector mediated by survivin promoter for the targeted gene therapy in cervical cancer.Methods: The total RNA was extracted from human peripheral blood lymphocytes(PBMCs) and was used as Trail PCR template,TRAIL gene with interleukin 2(IL-2) signal peptide gene was amplified by RT-PCR method using primers with IL-2 gene sequence in 5' end of primer,and the PCR products were cloned into the vector pGL-Basic/surp downstream survivin promoter to form an eukaryotic expression vector that was then transfected into Hela and normal hepatocyte(7702).Expression of TRAIL was identified by RT-PCR and western blot from transfected cell.Results: A 613 bp cDNA fragment was amplified from PBMCs RNA and was further conformed by sequencing as IL-2 and trail fusion gene which was cloned into pGL-Basic/surp vector downstream survivin promoter to form eukaryotic expression vector pGL-Basic/surp/TRAIL.Enzyme digestion showed the correct recombinant.The results of the RT-PCR and western blot showed that pGL-Basic/surp/TRAIL vector tranfected Hela cells successfully expressed TRAIL,while there was no TRAIL expression in 7702 cell.Conclusion: The successful construction of survivin promoter controlling TRAIL eukaryotic expression vector provides the possibility for effective gene therapy of cervical cancer.