恶性人脑胶质瘤的HSV-Tk/GCV自杀基因治疗系统的构建及应用研究

目的构建恶性人脑胶质瘤细胞株TJ899的单纯疱疹病毒的胸腺激酶/丙氧鸟苷（HSV-Tk/GCV）自杀基因治疗系统，探讨该体系在脑胶质瘤治疗中的作用。方法采用PCR、RT-PCR、融合PCR、酶切、连接等技术构建由自杀基因pcDNA3.1（-）CMVTK表达载体；用脂质体介导法将pCMVTK质粒转染进入TJ899恶性人脑胶质瘤细胞，并用M IT法检测感染后细胞对（GCV）的敏感性。结果pCMVTK质粒经测序证实含有TK目的基因；脂质体介导Pcmvtk质粒成功转染了TJ899细胞，并在体外实验研究中显示很好的治疗效果。结论建立了作用于恶性人脑胶质瘤细胞株TJ899的HSV-Tk/GCV自杀基因治疗系统，为脑胶质瘤的临床研究打下了基础。

Construction and clinical application of HSV Tk/GCV gene therapy system of malignant glioma

Objective To construct plasmid vector pcDNA3.1(-)CMVTK and evaluate the effects of Lipo-mediated in vitro gene transfer of pcDNA3.1(-)CMVTK in cell proliferation and anglo-genesis of malignant glioma cells TJ899.Methods We constructed the expression plasmid vector of pcDNA3.1(-)CMVTK with PCR,RT-PCR,confusion PCR,enzyme restriction,ligation and so on.Combined the Lipofection with suicide gene pcDNA3.1(-)CMVTK for transfection the TJ899 cells,MTT analysis was used to detect the cytotoxic effects of the pcDNA3.1(-)CMVTK /5-FC system.Result The plasmid vector contained the target TK gene which was confirmed by the sequencing.The Lipo-mediated pcDNA3.1(-) CMVTK were effectively transfered into TJ899 cells and showed high effective gene therapy in vitro.Conclusion We constructed plasmid vector pcDNA3.1(-)CMVTK gene therapy systems,which set up foundation of clinic therapy for malignant glioma.