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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/ml)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≥10μmol/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 study the killing effect of 5-FC / CD :: UPRT combined with gene therapy on C6 glioma cells. Methods The yCD :: UPRT fusion gene was amplified and the recombinant expression vector containing yCD :: UPRT gene was constructed. The vector was transfected into packaging cell PT67 and the recombinant virus was transfected into C6 glioma cells. The positive clones were screened and identified. MTT Method to detect the killing effect of different concentrations of 5-FC on CD :: UPRT transgenic cells. Results The full-length CD :: UPRT gene was amplified by PCR. The sequence was confirmed by sequencing. The recombinant plasmid pLXSN-yCD :: UPRT was double-digested with restriction enzyme and the vector was transfected into packaging cells. The virus (titer of 3.5 × 10 ~ 6CFU / ml) was transfected into C6 cells, and the positive clone C6-yCD :: UPRT cell line was obtained by screening. The results showed that the target gene was efficiently expressed in this cell line. When the final concentration of 5-FC was ≥10μmol / L, the cell proliferation of the experimental group was significantly different from that of the control group (P <0.01). The apoptotic bodies were observed under electron microscope after 5-FC treatment for 96h. Conclusion 5-FC / yCD :: UPRT combined with gene therapy strategy has obvious cytotoxic effect on C6 glioma cells.