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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。
Objective To investigate the killing effect of HSV-TK / GCV suicide gene system on human cervical carcinoma cell line Hela in vitro and in vivo and the bystander effect of HSV-TK / GCV suicide gene system. Methods The GINaTK vector was transfected into packaging cell PA317 by lipofection. Human cervical cancer cell Hela was infected with the virus supernatant to obtain Hela / TK cells with HSV-TK gene and used for in vitro and in vivo experiments respectively. In vitro, the sensitivity and bystander effect of Hela / TK cells to gonadotropin (GCV) were determined by MTT assay. In animal experiments, Hela / TK and Hela cells were injected subcutaneously in the right armpit of BALB / C mice at different ratios and then treated with GCV. The expression of HSV-TK gene in tumor tissue was detected by RT-PCR. Results Hela / TK cells transfected with HSV-TK gene showed stronger killing tumor effect than Hela cells. In vitro experiments showed that when Hela / TK cells accounted for 10% of mixed cells, low concentration (10μg.ml-1) of GCV can kill about 50% of tumor cells. In vivo experiments showed that GCV significantly inhibited the tumor formation of Hela / TK cells in BALB / C mice. After GCV treatment, the tumor volume in Hela / TK group and mixed cell group was 52.8% and 69.4% smaller than that in control group (all P <0.001). The mean survival of mice was also significantly longer (P <0.001). Conclusion The retrovirus can transfer HSV-TK gene into human cervical cancer cell Hela and get stable expression. The HSV-TK / GCV suicide gene system has killing effect on cervical cancer cell both in vitro and in vivo, and there is obvious bystander effect .