携带双调控自杀基因的重组腺相关病毒载体的构建及初步应用  

The Construction of Recombinant AAV Vector Mediated Doudle Regulatory Suicide Gene and Its Preliminary Application

作　　者：李子博[1] 曾赵军[2] 孙开胜[1] 

机构地区：[1]长沙医学院医学检验系,长沙410219 [2]中南大学生物科学与技术学院分子生物学研究中心,长沙410078

出　　处：《长沙医学院学报》2008年第2期12-16,共5页Journal of Changsha Medical University

基　　金：湖南省教育厅基金资助项目(项目编号:07C151)

摘　　要：目的:为构建携带受Tet-On以及VEGF启动子调控自杀基因的重组腺相关病毒载体。方法:用PCR方法扩增VEGF启动子,将其插入pAAV/TRE/HSVtk/Tet-On,形成重组载体pAAV/VEGF/ TRE/HSVtk/Tet-On质粒。进行病毒包装后得到了AAV/VEGF/TRE/HSVtk/Tet-On重组腺相关病毒。用重组腺相关病毒感染乳腺癌细胞株MCF-7和正常乳腺HBL-100细胞,用MTT法及RT-PCR检测在Dox诱导下,GCV对rAAV感染的MCF-7细胞和HBL-100细胞的杀伤作用以及HSVtk基因在MCF-7细胞内的表达情况。结果:在rAAV+Dox+GCV组,GCV对rAAV感染的MCF-7细胞的杀伤作用明显高于rAAV+Dox组,rAAV+Dox组以及HBL-100组,并且RT-PCR结果显示经Dox诱导HSVtk表达较明显。结论:成功构建了携带双调控自杀基因的重组腺相关病毒载体,该病毒载体能有效的感染乳腺癌细胞MCF-7,并能联合GCV治疗,抑制肿瘤细胞生长,而且具有靶向性。Objective To construct the recombinant AAV vector mediated regulatory suicide gene and elucidate value of its antitumor.Methods:VEGF promoter was inserted into theplasmid pAAV/ TRE/HSVtk/Tet-On and the recombinant plasmid pAAV/VEGF/TRE/HSVtk/Tet-On was constructed, and AAV/VEGF/TRE/HSVtk/Tet-On recombinant adeno-associated virus(rAAV)were then harvested. Purified rAAV was used to infect breast cancer cell strain MCF-7 and normal cell strain HBL-100,killing effects of GCV on MCF-7 cells infected with rAAV and expression HSVtk gene on the intra cells were detected using MTT method and RT-PCR assay under doxycycline(Dox)induction.Results:The viruses infected MCF-7 cell and showed that the mortality of MCF-7 cells in the rAAV+Dox+GCV group was obviously higher than that in rAAV+GCV group、rAAV+Dox group,as well as HBL-100 group(p<0.05), it suggests Dox-induction enhanced tumor killing effects of GCV.Conclusions The constructed rAAV/ VEGF/TRE/HSVtk/Tet-On can effective enhancely efficacy of GCV antitumor,inhibit the tumor cell growth,and the gene can specifically express at the targetted cell.

关 键 词：基因治疗 腺相关病毒 VEGF启动子 HSVtk基因 TET-ON 

分 类 号：R346[医药卫生—基础医学] R73-3