【摘要】 自杀基因治疗是恶性肿瘤基因治疗中最常用的途径之一 .以逆转录病毒载体———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 .更多还原

【Abstract】 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.更多还原