HSV-tk/GCV自杀基因系统对骨肉瘤细胞体外杀伤效应

目的：观察：HSV—tk／GCV系统对骨肉瘤细胞SAOS-2的体外杀伤作用．方法：构建tk基因逆转录表达载体pL(tk)SN，在lipofectamine2000介导下转染PA317包装细胞，G418筛选，直至出现抗性克隆，扩大培养，用NIH3T3测定病毒滴度，将重组病毒感染人骨肉瘤细胞SAOS-2，G418筛选出抗性克隆命名为SAOS／tk，以RT—PCR及Southern blot检测tk基因的整合及表达．观察tk基因修饰细胞生长状况及MTT法观察前药对tk基因修饰骨肉瘤细胞的杀伤效应．结果：RT—PCR及Southern blot分析证明tk基因在人骨肉瘤细胞系SAOS-2中有整合及表达；SAOS／tk与未转基因的原肿瘤细胞相比，两者生长速度及形态结构无明显差别；在前药敏感性试验中，GCV对SAOS／tk细胞的杀伤作用十分明显，半数致死量约为1mg／L，而亲本细胞SAOS-2和空白质粒转染SAOS／0细胞，半数致死量大于100mg／L．tk基因修饰细胞与不同比例亲本细胞混合后经GCV治疗，SAOS／tk占混合细胞总数的20％时即可杀伤约50％的混合培养细胞.结论：人骨肉瘤细胞系SAOS-2对HSV—tk／GCV系统高度敏感，并且有明显的旁观者效应，可望成为骨肉瘤基因治疗的新途径．

Killing effect of GCV combined with suicide gene HSV-tk on human osteosarcoma cells in vitro

AIM: To investigate the killing effect of GCV combined with suicide gene HSV tk on SAOS 2 cells in vitro . METHODS: Retroviral expressing vector pL(tk)SN was generated by recombinant DNA technique. SAOS 2 was infected by the recombinant retrovirus expressing tk gene. The positive clones were obtained by G418 selection and termed as SAOS/tk. The integration and expression of tk gene in SAOS 2 cells were identified by RT PCR and Southern blot. The growth state and prodrugs' killing effect of tk gene modified cells were investigated with the expression of tk gene and antitumor effect on SAOS 2 cells. RESULTS: RT PCR and Southern blot analysis confirmed the integration and expression of tk gene in retrovirus transduced SAOS 2 cells. There was no significant difference in cell proliferation between the SAOS/tk and SAOS 2. SAOS/tk showed high sensitivity to GCV and bystander effect in vitro . CONCLUSION: The osteosarcoma cells SAOS 2 are sensitive to HSV tk/GCV system and have significant bystander effect, which might have therapeutic potentials for osteosarcoma.