HSV-TK/GCV自杀基因系统治疗宫颈癌的研究

目的探讨HSV-TK/GCV自杀基因系统对人宫颈癌细胞系Hela体外及体内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PA317.取病毒上清液感染人宫颈癌细胞Hela,得到带有HSV-TK基因的Hela/TK细胞,并将其分别用于体外和体内实验.结果载体HSV-TK导入了PA317细胞.体外实验结果显示,当Hela/TK细胞数占混合细胞10%时,低浓度(10μg/ml)的GCV就可将50%左右的肿瘤细胞杀死.体内实验结果显示GCV可明显抑制Hela/TK细胞在BALB/C小鼠体内的肿瘤形成.经GCV治疗后,肿瘤体积分别较对照组肿瘤体积缩小约11.1%、30.6%和47.2%(均P＜0.001);RT-PCR检测HSV-TK基因在肿瘤组织中有表达;实验组肿瘤组织与对照组相比存在明显的病理学改变.结论逆转录病毒可介导HSV-TK基因转入人宫颈癌细胞Hela并获稳定表达,HSV-TK/GCV自杀基因系统在体内外对宫颈癌细胞均有杀伤作用,且存在明显的旁观者效应.

A study of the RV - HSV- TK/GCV suicide gene therapy system in cervix cancer

Objective: To study in vitro and in vivo killing effect and the bystander effect of HSV-TK/GCV suicide gene system on human Cervix cancer cells Hela.Methods: GINaTK retroviral vector containing HSV-TK gene was transduced into PA317 packaging cell by lipofectin.Human Cervix cancer cell line Hela was infected by high titer viral supernatant.PCR was resorted to demonstrate the successful transduction of the HSV-TK gene.Hela/TK cells and Hela cells were used in in vitro and in vivo study.Results: PA317 cells were transfected successfully with HSV-TK gene by lipofectin(named PA317/TK).Experimental results revealed that stable virus producing cell line was established and Hela/TK cells expressing the HSV-TK gene were obtained successfully.In in vitro study,when the ratio of Hela/TK cells reached 10%,the tumor cell-killing proportion was almost 50 %.I In in vivo study,GCV could suppress tumor formation of the Hela/TK cells.After mice treated with GCV,the median tumor volume of mice implanted with Hela/TK+Hela(1:9)group,Hela/TK+Hela(1:1)and Hela/TK cells was respectively decreased to 88.9%(P<0.001) and 69.4% and 52.8% compared with the control tumors.The expression of HSV-TK gene was detected by RT-PCR.Tumors treated with GCV revealed different histopathological features compared with the control tumors.Conclusion:The test showed that the HSV-TK gene can be transducted into Human Cervix cancer line Hela under the mediation of retrovirus and be stable expressed,HSV-TK/GCV suicide gene therapy system could improve the antitumoral efficiency.The bystander effect could be observated in HSV-TK/GCV system in in vitro and in vivo.