受强力霉素调节的自杀基因表达载体系统的构建和在乳腺癌细胞株(MCF-7)的表达

自杀基因治疗是恶性肿瘤基因治疗中最常用的途径之一，以递转录病毒载体-pRevTRE为基础进行载体构建，首先使用PCR技术对单纯疱疹病毒胸苷激酶基因（HSVtk)进行扩增，将HSVtk基因插入到pRe-vTRE，形成重组载体pRevTRE/HSVtk，用磷酸钙共沉淀法，经过两轮转染，分别将pRevTRE/HSVtk和pRevTet-On质粒导入乳腺癌细胞株（MCF-7）经过潮霉素B（HygromycinB)和G418筛选，建立了一株稳定的受四环素衍生物-强力霉素（Doxycycline,Dox)调控，表达HSVtk基因产物的人乳腺癌细胞株MCF/TRE/tk/Tet-On,HSVtk基因表达产物可以将无毒性的药物前体Ganciclovir(GCV)转变成一种有毒的代谢产物，从而杀死乳腺癌细胞株（MCF-7），达到基因治疗的目的。

The Construction of Suicide Gene Expression Vector Controlled by Tet-On and the Transfection of Human Breast Cancer Cell Line (MCF-7)

Suicide gene therapy is one of the choices in the cancer gene therapy research. RevTet-On gene expression system was used to deliver the suicide gene HSVtk to human breast cancer cell line MCF-7 and the tk gene expression was controlled by tetracycline derivative, doxycycline (Dox). HSVtk gene was inserted into the plasmid pRevTRE and the recombinant retroviral vector pRevTRE/HSVtk was constructed. Suicide gene HSVtk was expressed in the presence of Dox. Two transfections of pRevTRE/HSVtk and pRevTet-On were performed for MCF-7 cell line by using modified calcium phosphateco-precipitation method respectively, followed by Hygromycin B and G418. Tet-On MCF-7 cell line stably expressing tet-regulated tk gene was established. This cell line was named as MCF/tk/TRE/Tet-On and HSVtk gene expression was under the control of Dox.