携带靶向性可调控自杀基因的重组相关腺病毒载体的构建及其活性检测

The Construction of Recombinant AAV Vector Mediated Double Regulatory Suicide Gene and the Detection of its Activity

目的:构建携带受Tet-On以及VEGF启动子双调控自杀基因HSVtk的重组腺相关病毒载体,研究其在乳腺癌细胞MCF-7中的可调控表达。方法:用PCR方法扩增VEGF启动子,将其插入pAAV/TRE/HSVtk/Tet-On中形成重组载体pAAV/VEGF/TRE/HSVtk/Tet-On质粒。进行病毒包装后得到了rAAV/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 adeno-associated virus （rAAV） vector containing suicide gene HSVtk controlled by Tet-On gene regulation system and VEGF promoter and to detect the expression of HSVtk in the MCF-7 cells. Methods： VEGF promoter was inserted into the plasmid pAAV/TRE/HSVtk/Tet-On and the recombinant plasmid pAAV/VEGF/TRE/HSVtk/Tet-On was constructed ,and rAAV were then harvested. Purified rAAV was used to infect breast cancer cell strain MCF-7 and normal cell sWain HBL-100, killing effects of GCV on MCF-7 cells infected with rAAV and expression of HSVtk gene in the infected MCF-7 cells were detected using MTT method and RT-PCR assay under doxycycline（Dox） induction. Results： The viruses infected MCF-7 cell 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） and HSVtk gene express obviously in MCF-7 cells under Dox induction,which suggests Dox-induction enhanced tumor killing effects of GCV and expression of HSVtk gene. Conclusions： The constructed rAAV/VEGF/TRE/HSVtk/Tet-On can effective enhance efficacy ofGCV antitumor, inhibit the tumor cell growth, and the gene can specifically express at the turnor cell.