RV-HSV-TK/GCV自杀基因系统治疗宫颈癌的实验研究

目的探讨单纯疱疹病毒胸苷激酶/丙氧鸟苷(HSV-TK/GCV)自杀基因系统对人宫颈癌细胞系Hela体外及体内的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.体外实验中,Hela/TK细胞对丙氧鸟苷(GCV)的敏感性和旁观者效应采用四甲基偶氮唑盐(MTT)法测定.动物试验中分别在BALB/C小鼠的右腋窝皮下按不同比例注射Hela/TK细胞和Hela细胞,然后给予GCV治疗.逆转录聚合酶链反应(RT-PCR)检测肿瘤组织中HSV-TK基因的表达情况.结果转染HSV-TK基因的Hela/TK细胞表现出比亲本Hela细胞更强的杀伤肿瘤效应.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-1)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,Hela/TK组和混合细胞组肿瘤体积分别较对照组肿瘤体积缩小52.8%和69.4%(均P＜0.001).小鼠的平均生存期也显著延长(P＜0.001).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.

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

Objective To studyin vitroandin vivokilling effect andthe bystander effect of HSV-TK/GCVsuicide gene systemon cervix cancer cell line Hela.Methods GINaTKretroviral vector containing HSV-TKgene wastransducedinto PA317 packaging cellby lipofectin.Cervix cancer cell line Hela wasinfected by hightiter viral supernatant.Hela/TKcells and Hela cells were usedininvit-roandin vivostudy.Inin vitrostudy,sensitivity of Hela/TKcells to GCVand bystander effect were observed by MTTtest.Inin vivostudy,Hela/TKcells and Hela cells were injected s.c.intoflanking BALB/C mice,GCVwas administratedi.p.,RT-PCR was appliedto detect the expression of HSV-TKgene.Statistical analysis of the data was performed by usingthe ANOVA(analysis of variance).Re-sults The Hela cells transfered with GINaTKgene displayed a higher antitumor effect than parent cells.Inin vitrostudy,whenthe ra-tio of Hela/TKcells reached 10 %,thetumor cell-killing proportion was 50 %.Inin vivostudy,all mice developedtumorsin7 days af-ter tumor cells implanted,the ratio of tumors formation was 100 %.GCVcould suppress tumor formation of the Hela/TKcells.Aftermice treated with GCV,the median tumor volume of mice implanted with Hela/TKcells and mice with cells mixed was decreased to52.8 %(P<0.001) and 69.4 %(P<0.001) respectively compared with the control tumors.Their median survival was significantlyprolonged(P<0.001).Conclusion The test showedthat the HSV-TKgene can be transductedinto cervix cancer cell Hela under themediation of retrovirus and be stable expressed,HSV-TK/GCVsuicide genetherapysystemcouldimprovethe antitumoral efficiency.Thebystander effect could be observatedin HSV-TK/GCVsysteminin vitroandin vivo.