摘要
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 In order to study the killing effect and the by stander effect of retrovirus mediated herpes simplex virus thymidine kinase(HSV TK)gene combined with ganciclovir(GCV) on mice breast carcinoma cells.Methods GINaTK retroviral vector containing HSV TK gene was transduced into PA317 retroviral packaging cells by lipofectin,PA317 cells transfected with HSV TK gene were then screened by G418 medium,and resistant clones(named PA317/TK) were identified by PCR.Mice breast carcinoma cell line MA782/5S 8102 was infected by the high titer viral supernatant.MA782/5S 8102/TK cells and MA782/5S 8102 cells were mixed in different ratio and cultured in 96 well plates.After culture for 24 hours,10 μg/ml GCV was added into half of the wells,the others as controls.4 days later,viability of the cells was obtained by MTT test.Results Experimental results revealed that stable virus producing cell line was established after GINaTK retroviral vector was transferred into PA317,and MA782/5S 8102/TK cells expressing the HSV TK gene were obtained successfully.As showing by mixed culture of MA782/5S 8102/TK cells and MA782/5S 8102 cells in different ratio,when the percentage of MA782/5S 8102/TK cells reached 10%,the killing rate of tumor cells was almost 50%.Conclusion The HSV TK gene can be stable expressed in mice breast cancer line MA782/5S 8102 under the mediation of retrovirus,and the bystander effect of HSV TK/GCV system can improve the killing effect of cancer cells.
出处
《实用癌症杂志》
2003年第2期122-125,共4页
The Practical Journal of Cancer
基金
国家自然科学基金 (批准号 :30 0 70 2 2 9)
