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目的 探讨逆转录病毒 (RV )载体介导的单纯疱疹病毒胸苷激酶 (HSV -TK )基因转染小鼠乳腺癌细胞 ,联合抗病毒药物丙氧鸟苷 (GCV )对小鼠乳腺癌细胞系MA 782 /5S -810 2体外杀伤作用及其产生的旁观者效应。方法 采用脂质体转染法将GINaTK载体转入包装细胞PA3 17、G 418筛选抗性克隆 ,采用PCR方法检测HSV -TK基因整合情况。取滴度最高的病毒上清液感染小鼠乳腺癌细胞MA 782 /5S -810 2 ,经G 418筛选后 ,得到带有HSV -TK基因的MA 782 /5S -810 2 /TK细胞 ;将MA 782 /5S -810 2和MA 782 /5S -810 2 /TK细胞按不同比例混合培养后 ,给予 10 μg/mlGCV ,4天后采用MTT法计算细胞成活率 (SR ) ,观察旁观者效应。结果 GINaTK载体成功转入包装细胞PA 3 17,病毒滴度为 4.0× 10 4cfu/ml。用病毒上清液感染MA 782 /5S -810 2 ,成功得到了表达HSV -TK基因的MA 782 /5S -810 2 /TK细胞。混合培养结果显示 ,当MA782 /5S -810 2 /TK细胞数占混合细胞数 10 %时 ,低浓度 (10 μg/ml)的GCV就可将 5 0 %左右的肿瘤细胞杀死。 结论 逆转录病毒可介导HSV -TK基因转入小鼠乳腺癌细胞MA782 /5S -810 2并获稳定表达 ,RV -HSV -TK /GCV系统杀伤癌细胞存在旁观者效应。
OBJECTIVE: To investigate the effect of retroviral (RV) vector-mediated HSV-TK gene transfer on mouse breast cancer cells in combination with antiviral drug ganciclovir (GCV) on mouse breast cancer cell lines In vitro killing of MA 782 / 5S -810 2 and its bystander effect. Methods The GINaTK vector was transfected into PA3 17 packaging cells by lipofection method. The resistant clones were screened by G 418, and the integration of HSV-TK gene was detected by PCR. The virus titer with the highest titer was inoculated into MA 782 / 5S -810 2 mouse breast cancer cells and screened by G 418 to obtain MA 782 / 5S -810 2 / TK cells with HSV-TK gene. MA The mixed culture of 782 / 5S -810 2 and MA 782 / 5S -810 2 / TK cells in different proportions was given 10 μg / ml GCV. After 4 days, the cell viability (SR) was calculated by MTT assay. Results The GINaTK vector was successfully transfected into packaging cells PA 3 17 with a virus titer of 4.0 × 10 4 cfu / ml. MA 782 / 5S -810 2 / TK cells expressing the HSV-TK gene were successfully obtained by infecting MA 782 / 5S -810 2 with the virus supernatant. The mixed culture results showed that about 50% of the tumor cells could be killed by GCV at a low concentration (10 μg / ml) when MA782 / 5S-8102 / TK cells accounted for 10% of the mixed cells. Conclusion The retroviral vector transfected HSV-TK gene into mouse mammary carcinoma cell line MA782 / 5S -810 2 was stably expressed. The bystander effect was observed in RV-HSV-TK / GCV system.