HSV-tk/GCV系统对小鼠肝癌细胞体外杀伤效应的研究

目的:研究HSV-tk/GCV系统对小鼠肝癌细胞的体外杀伤作用.方法:利用重组DNA技术,将HSV-tk基因亚克隆至逆转录病毒载体(pLNCTK)中,在LipofectAMINE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NTH3T3细胞测定病毒滴度,将重组逆转录病毒感染小鼠肝癌细胞MM45T.Li,G418筛选,直至出现抗性克隆,挑取阳性克隆,扩大培养,命名为MM45T.Li/TK.PCR,RT-PCR对HSV-tk基因修饰的MM45T.Li细胞进行鉴定,然后观察CCV对MM45T.Li/TK和不同比例TK~ 与TK~-混合细胞的杀伤作用.结果:重组逆转录病毒滴度为5×10~5CFU/ml,PCR及RT-PCR分析证明HSV-tk基因已整合至MM45T.Li细胞基因组中,并在mRNA水平上的表达.MM45T.Li/TK与未转基因的原肿瘤细胞,二者的生长速度与形态结构无明显差别.MM45T.Li/TK细胞对CCV高度敏感,即低浓度GCV(1mg/L)处理MM45T.Li/TK细胞3d,即可将其杀死.旁观者效应显示将25%MM45T.Li/TK细胞与75%MM45T.Li混合,发现90%以上的细胞被杀死.结论:小鼠肝癌细胞对HSV-tk/CCV系统高度敏感,并具有明显的旁观者效应.

Study on Killing Effects of HSV-tk/GCV System on Murine Liver Cancer Cells in vitro

Objective: To investigate the killing effects of HSV-tk/GCV system on murine liver cancer cells in vitro. Methods: HSV-tk gene was cloned into retroviral vector (pLNCTK) by recombinant DNA technology. After packaging with PA317 cell line, murine liver cancer cell line MM45T.Li was infected by the recombinant retrovirus. The positive clones were obtained after G418 selection and it was termed MM45T. Li/TK. The integration and expression of tk gene in MM45T. Li/TK cells were identified by PCR and RT-PCR. The killing effects of GCV on TK cell alone or a mixture of TK and TK~(-) cells in different ratio were observed. Results: (1) PCR and RT-PCR analysis confirmed integration of HSV-tk gene in positive clone, and expression of HSV-tk gene at mRNA level. (2) There was no significant difference in cell proliferation or morphology between the MM45T. Li/TK and MM45T. Li. (3) The cytotoxicity of GCV in MM45T. Li/TK cells was 1 000 fold higher sensitive than that in parental MM45T.U cells. (4) When MM45T.Li/TK cells were mixed with parental MM45T. Li cells at a ratio of 25:75, significant bystander effect was observed in vitro after they were treated with nontoxic GCV. Conclusions: These results suggested that murine liver cancer cells were sensitive to HSV-tk/GCV system and had significant bystander effects.