论文部分内容阅读
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 a recombinant adeno-associated virus vector carrying suicide genes regulated by Tet-On and VEGF promoters. Methods: The VEGF promoter was amplified by PCR and inserted into pAAV / TRE / HSVtk / Tet-On to construct the recombinant vector pAAV / VEGF / TRE / HSVtk / Tet-On. AAV / VEGF / TRE / HSVtk / Tet-On recombinant adeno-associated virus was obtained after virus packaging. The recombinant adeno-associated virus was used to infect MCF-7 breast cancer cells and normal breast HBL-100 cells. MTT assay and RT-PCR were used to detect the killing effect of GCV on rAAV-infected MCF-7 cells and HBL-100 cells induced by Dox Role and HSVtk gene expression in MCF-7 cells. Results: The killing effect of GCV on rAAV-infected MCF-7 cells in rAAV + Dox + GCV group was significantly higher than that in rAAV + Dox group, rAAV + Dox group and HBL-100 group. RT-PCR results showed that HSVtk The expression is more obvious. CONCLUSION: A recombinant adeno-associated virus vector carrying double-regulated suicide gene has been successfully constructed. The vector can effectively infect breast cancer cell line MCF-7 and can be combined with GCV to inhibit the growth of tumor cells.