论文部分内容阅读
探讨逆转录病毒 (retrovirus ,RV )载体介导的单纯疱疹病毒胸苷激酶 (HSV1 tk )基因转染 ,联合抗病毒药物核苷类似物羟甲基无环鸟苷 (ganciclovir,GCV )对人卵巢上皮癌细胞系TYK细胞杀伤过程中所产生的旁观者效应。采用脂质体介导法将PLNTK5质粒转入包装细胞PA317后 ,以滴度最高的PA317病毒上清液感染TYK细胞 ,遗传霉素G418筛选后 ,得到带有HSV1 tk基因的TYK细胞。将细胞按不同比例混合培养后 ,给予 10 μg/mlGCV ,4d后用MTT法计算细胞存活率 ,观察旁观者效应。PLNTK5质粒成功转入PA317细胞 ,病毒滴度最高者为 6× 10 5cfu/ml。用病毒上清液感染TYK细胞 ,成功地得到了表达HSV1 tk的卵巢癌细胞株TYK/tk。混合培养结果显示 ,TYK/tk细胞占混合细胞 10 %时 ,低浓度的GCV就可使一半以上的细胞杀死 ,证明了HSV1 tk/GCV治疗方法存在着旁观者效应。逆转录病毒可介导HSV1 tk基因转入TYK细胞并获稳定表达 ,HSV tk/GCV系统存在旁观者效应。
To investigate the effect of retrovirus (RV) vector-mediated transfection of herpes simplex virus thymidine kinase (HSV1 tk) gene and ganciclovir (GCV), an antiviral nucleoside analogue, The bystander effect of TYK cell killing in epithelial cancer cell line. The TYK cells were infected with the PA317 virus supernatant with the highest titer after the PLNTK5 plasmid was transformed into the packaging cell PA317 by liposome-mediated method. The CHO cells transfected with the tk gene were selected by G418 and TYK cells with HSV1 tk gene were obtained. The cells were mixed at different ratios and then given 10 μg / ml GCV. After 4 days, the cell viability was calculated by MTT method and the bystander effect was observed. The PLNTK5 plasmid was successfully transfected into PA317 cells with the highest virus titer of 6 × 10 5 cfu / ml. TYK / tk was successfully obtained in TYK cells infected with virus supernatant. Mixed cultures showed that TYK / tk cells killed more than half the cells at low concentrations of GCV at 10% of the mixed cells, demonstrating the bystander effect of the HSV1 tk / GCV treatment. The retrovirus can transduce the HSV1 tk gene into TYK cells and get stably expressed. The HSV tk / GCV system has bystander effect.