逆转录病毒载体介导中国单疱病毒TK基因转导脑胶质瘤细胞

Transfer thymidine kinase gene of Chinese herpes simplex virus I mediated by retrovirus to glioma cells in vitra

目的：研究载中国单疱病毒胸苷激酶基因逆转录病毒重组体（ＲＶ－ＨＳＶ－ＴＫｃ）和羟甲基无环鸟苷（ＧＣＶ）体外转染和杀伤脑胶质瘤效果，探索应用该系统基因治疗脑胶质瘤。方法：ＲＶ－ＨＳＶ－ＴＫｃ重组体病毒感染胶质瘤细胞，Ｇ４１８筛选转染阳性胶质瘤细胞克隆。Ｎｏｒｔｈｅｒｎ杂交检测抗Ｇ４１８胶质瘤细胞中的ＴＫｃ基因表达。药物敏感实验观察ＴＫｃ胶质瘤细胞对ＧＣＶ的毒性反应。结果：１．０ｍｇ／ｍｌＧ４１８筛选１０～１４天获抗性胶质瘤细胞克隆；Ｎｏｒｔｈｅｒｎ杂交显示抗Ｇ４１８细胞ＴＫｃ基因明显表达；ＴＫｃ胶质瘤细胞对ＧＣＶ高度敏感，其ＥＤ５０为：Ｃ６ＴＫｃ０．０２μｇ／ｍｌ，Ｕ２５１ＴＫｃ０．００６μｇ／ｍｌ，Ｕ８７ＴＫｃ０．００４μｇ／ｍｌ，ＴＫｃ胶质瘤细胞对ＧＣＶ的敏感性是非转基因胶质瘤细胞的５００倍以上。结论：载中国ＴＫｃ基因逆转录病毒重组体可有效转染脑胶质瘤细胞并使其对ＧＣＶ高度敏感，该系统可望用于脑胶质瘤的基因治疗。

Objective: To apply gene therapy to malignant gliomas with RV HSV TK/GCV system in China, we performed the study of retrovirus recombinants with thymidine kinase gene of Chinese herpes simplex virus I transfecting glioma cells in vitra and killing the cells with TKc gene in ganciclovir (GCV). Methods: In G418 medium, the clones of transfected glioma cells were selected after infection of cells in virus of RV HSV TKc. Northern blotting analysis was used to check TKc gene expression in G418 resistant glioma cells. Cytotoxic experiment was performed on GCV to glioma cells with TKc gene. Results: The clones of G418 resistant cells with TKc gene were obtained in 1 0mg/ml G418 medium for 10～14 days. Significantly TKc gene expression in these resistant glioma cells in G418 was found with Northern blot. The cells with TKc gene were of high sensitivity to GCV in cytotoxic experiment, in which ED50 of TKc glioma cells to GCV shown C6TKc 0 02 μg/ml; U87TKc 0 004μg/ml, U251TKc 0 006μg/ml that were over 500 folds of glioma cells without TKc gene. Conclusion: Chinese system of retrovirus recombinant with HSV TKc gene is of reliable for cytotoxic effect of glioma cells in vitro. It can be used potentially in gene therapy of malignant brain tumors in future.