真菌源性CD::UPRT联合基因治疗脑胶质瘤的实验研究  

Efficacy of 5-FC/yeast CD::UPRT combined gene therapy for malignant glioma

作　　者：胡卫星[1] 封林森[1] 卢春[1] 唐桂霞[1] 史德志[1] 贾雪梅[1] 李立新[1] 魏栋[1] 

机构地区：[1]南京医科大学第一附属医院神经外科,210029

出　　处：《中华神经外科杂志》2006年第6期377-379,共3页Chinese Journal of Neurosurgery

基　　金：江苏省医学重点人才工程(135工程)资助(编号RC2002075)

摘　　要：目的 研究5-FC／CD：：UPRT联合基因治疗策略对胶质瘤细胞C6的杀伤效应。方法 扩增yCD：：UPRT融合基因并构建含yCD：：UPRT基因的重组表达载体；载体转染包装细胞PT67，所获重组病毒转染胶质瘤细胞C6，筛选并鉴定阳性转基因克隆；用MTT法检测不同浓度5-FC对CD：：UPRT转基因细胞的杀伤效应。结果 PCR法扩增出全长CD：：UPRT基因，经测序证实序列正确，重组逆转录病毒表达载体pLXSN—yCD：：UPRT经双酶切获目的条带，载体转染包装细胞获重组逆转录病毒（滴度达3．5×10^6CFU／m1）并转染C6，经筛选获得转基因阳性克隆C6-yCD：：UPRT细胞株，检测显示该细胞株有效表达目的基因。当5-FC终浓度≥10p,mol／L时，实验组与对照组的细胞增殖力出现显著差异（P〈0．01），5-FC作用96h后电镜观察到凋亡小体。结论 5-FC／yCD：：UPRT联合基因治疗策略对胶质瘤细胞C6有明显的杀伤作用。Objective To investigate the efficacy of 5-FC/yCD：： UPRT combined gene therapy on malignant glioma C6 cells in vitro. Methods PCR amplified yCD：： UPRT genes and reconstructed expressing retroviral vectors, pLXSN-yCD ： ： UPRT, were constructed. Reconstructed yCD ： ： UPRT-retrovirus were packaged in PT67 cell, and then infected malignant glioma cells, C6. The reconstructed C6-yCD： ： UPRT cell line was selected by G418. Finally, the toxicity efficacy of 5-FC on C6-yCD：：UPRT cells was checked by electron-microscope and MTT assays. Results The sequence of amplified yCD： ： UPRT gene was confirmed. The yCD： ：UPRT bands from pLXSN-yCD： ： UPRT were obtained on PCR and RT-PCR. The titer of yCD： ：UPRT-retrovirus was 3.5×10^6CFU/ml. C6-yCD： ：UPRT cell line was established with the same growth characters as C6. yCD： ：UPRT gene was expressed efficiently by the cells. Electron-microscope showed apoptosis bodies in the cells, which were cultured with 5-FC 50μg/ml for 96 hrs. MTT assay showed that the growth ability of the cell was sharply suppressed when 5-FC concentration was ≥v10μmol/L（ P 〈 0.01 ）. Conclusions 5-FC/yCD：： UPRT combined gene therapy has a significant killing efficacy on malignant glioma C6 cells.

关 键 词：基因治疗自杀基因 CD::UPRT 逆转录病毒 胶质瘤细胞 

分 类 号：R739.4[医药卫生—肿瘤]