为研究阳离子脂质体介导HSV-tk自杀基因系统的体内外抗肝癌作用,本课题用基因重组技术构建了含HSV-tk基因的重组逆转录病毒载体,命名为pLXT。用Lipofectin介导转染人肝癌细胞系SMMC-7721,经G418筛选抗性克隆及流式细胞仪分析,获得了持续表达HSV-tk的克隆细胞SM/tk。~3H-TdR掺入法测定表明HSV-tk/ACV系统对SM/tk细胞具有强杀伤力。将Lipofectin-pLXT复合物直接注射于鼠肝癌细胞(H22)实体瘤内,ACV治疗后监测抑瘤率,发现脂质体介导pLXT、ACV治疗组及裸pLXT注射、ACV治疗组平均瘤体积均显著小于其它六组对照组(P<0.001,P<0.05),前两者比较亦有显著差异(P<0.02)。空载体转染组和阴性对照组平均瘤体积及平均瘤重均无显著差异(P>0.05)。这些结果表明阳离子脂质体介导重组逆转录病毒载体基因转染的方法简便、安全、有效,且可使目的基因获得持续表达;在体内HSV-tk/ACV系统具有强的细胞毒作用,提示存在着强的旁观者效应。此外,瘤体内直接注射裸HSV-tk基因的治疗也有效。

In order to investigate the effect of anti-hepato-cellular carcinoma of HSV-tk suicide gene system, we constructed the HSV - tk recombinant retroviral vector pLXT. SMMC - 7721 hepatocellular carconoma cells more transfected with pLXT by lipofectin were obtained by subsequent G418 screen. 3H-TdR incorparation assay showed that HSV - tk/ACV had strong cytotoxic effect on HSV - tk gene transfected tumor cells. Lipofectin pLXT complex was directly injected into murine H22 hepatoma tissue, followed by delivery of ACV prodrup, and it was found that the tumor growth masses more greatly reduced. Animals treated with Liptk ACV and tk ACV had an apparent reduction of tumor size as compared with the animals in other six groups ( P < 0.001, P < 0.05). In addition, there was a significant reduction of tumor size between animals treated with Liptk ACV and tk ACV ( P < 0.02). No significant difference was observed in terms of growth rate of tumors among other six control groups. These results indicate the approach that the recombinant retroviral vector was transduced by cationic liposome is simple, safe and efficient, and could make HSV - tk gene acquire persistence of expression in HCC cells. HSV - tk/ACV system showed strong cytotoxic effect, which suggested to has a strong bystander effect in vivo. In addition, it is efficient that nude HSV-tk gene is directly injected into hepatocellular carcinoma tissue.