逆转录病毒介导的HSV_1-tk基因疗法对雄激素非依赖性前列腺癌细胞系的杀伤效应

Retrovirus-mediated HSV_1-tk gene therapy for androgen-independent prostate cancer

目的 探讨逆转录病毒载体介导单纯疱疹病毒Ⅰ型胸苷激酶 (HSV1 tk)结合环氧鸟苷(GCV) ,对雄激素非依赖性前列腺癌 (C4 2、PC3 )的治疗效果。 方法 通过DNA重组技术构建含tk基因的逆转录病毒载体 ,经转染包装细胞系PA3 17,选择出高病毒滴度的生产细胞系 (VPC) ,PCR、RT PCR检测VPC内HSV1 tk基因的整合和表达。VPC与癌细胞按不同比例混合培养 ,以硫基罗丹明B(SRB)法测定GCV治疗后癌细胞生长情况。 结果 经阳性克隆筛选 ,得到高滴度并能稳定表达HSV1 tk基因的VPC细胞。逆转录病毒载体介导的HSV1 tk基因对雄激素非依赖性前列腺癌C4 2、PC3均具有杀伤效应 ,光镜下没有发现凋亡小体 ;经比较 ,HSV1 tk基因对PC3的治疗效果最好 ,差别具有显著性意义。 结论 逆转录病毒载体介导的HSV1 tk基因对雄激素非依赖性前列腺癌细胞有杀伤作用 。

Objective To use HSV 1 tk (herpes simplex virus type Ⅰthymidine kinase)/GCV (ganciclovir) in androgen independent prostate cancer cells(C4 2,PC3) in vitro in order to provide useful basis for clinical use. Methods HSV 1 tk gene was ligated to a pN 2A retroviral vector. Recombinant DNA molecules being introduced into a packaging cell line PA317,the high titer virus producer cells (VPC) were screened.The integration and expression of HSV 1 tk gene in VPC was observed by PCR and RT PCR. VPC was co cultured with these cancer cells in the light of 1∶1,1∶2,1∶4,1∶8.Cell viability (cytotoxicity) was assessed by SRB (sulforhodamine B protein dye binding)after the first day,the third day,the fifth and the seventh day. Results The highest titer VPC producing HSV 1 tk gene was isolated. Retrovirus mediated HSV 1 tk gene therapy was effective and active against such prostate cancer cells.The best one was co culture of VPC and cancer cells at 1∶1 and the fifth day followed by GCV. Compared with C4 2, PC3 decreased remarkably.The activation of apoptosis and other ways failed to be found. Conclusions Retrovirus mediated HSV 1 tk gene therapy in Vitro directly killed the tumor cells by cytolytic activity.