可调控重组腺相关病毒对MCF-7细胞基因转染效率及表达的研究

 目的 探讨Tet调控下自杀基因HSVtk重组腺相关病毒载体（rAAV/HSVtk/Tet-On）对人乳腺癌细胞株（MCF-7）的作用。方法 将HSVtk和Tet-On基因分别定向克隆入腺相关病毒质粒pAAV MCS中,构建重组腺相关病毒载体pAAV/TRE/HSVtk/Tet-On,与辅助质粒pAAV-RC、pHelper和包装细胞293进行包装、纯化。用β半乳糖苷酶原位染色法检测报告基因rAAV/LacZ在MCF-7中的表达,计算其基因转染效率。采用斑点杂交、RT-PCR检测MCF-7细胞基因组中HSVtk基因整合、病毒滴度及其表达。结果 rAAV/HSVtk/Tet-On病毒滴度为2.38×10^11particle/ml,LacZ基因在感染的MCF-7中能持续表达,感染效率为20%～30%。纯化后的重组腺相关病毒感染MCF-7后,能将目的基因转移到靶细胞中,并在Dox诱导下,使GCV对rAAV感染的MCF-7细胞具有明显的杀伤作用。结论 HSVtk/Tet-On自杀基因调控系统可通过重组腺相关病毒载体成功转染人乳腺癌细胞株MCF-7,使其HSVtkmRNA表达增加,在Dox诱导下,GCV对rAAV（≥10⁴v.p/cell）感染的MCF-7细胞具有明显的杀伤作用。

Gene Transfection Efficiency and Expression of Regulatory Recombinant Adeno-Associated Virus Gene Transfer through Direct Infection on MCF-7 Cells

Objective To explore the treatment of human breast cancer by adeno-associated virus (AAV) expressing HSVtk gene under the regulation of Tet regulatory system, the recombinant AAV-2 were transduced into MCF-7 cell line. Methods The Tet-On and HSVtk gene was respectively inserted into the AAV cassettes(pAAV MCS),then rAAV/HSVtk/Tet-On were generated by co-transfection 293 cells with other plasmids(pAAV-RC、pHelper). The viral titer of rAAV/HSVtk/Tet-On and its expression were measured by β-galactosidase staini...