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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.
Objective: To study the killing effect of HSV-tk/GCV system on mouse hepatoma cells in vitro. METHODS: The recombinant HSV-tk gene was subcloned into retroviral vector (pLNCTK) using recombinant DNA technology. Under the guidance of LipofectAMINE, The cells were stained with PA317 and screened with G418 until resistant colonies appeared. The culture was expanded. The virus titer was determined using NTH3T3 cells. The recombinant retrovirus was infected with mouse hepatoma cells MM45T.Li, G418 and screened until resistant clones appeared. Positive clones, expanded culture, named MM45T.Li/TK.PCR, RT-PCR identification of HSV-tk gene modified MM45T.Li cells, and then observe the CCV of MM45T.Li/TK and different ratios of TK~+ and TK Killing effect of ~-mixed cells. Results: The titer of recombinant retrovirus was 5×10~5 CFU/ml. PCR and RT-PCR analysis confirmed that HSV-tk gene was integrated into the genome of MM45T.Li cell and at mRNA level. The expression of MM45T.Li/TK and untransformed primary tumor cells showed no significant difference in growth rate and morphological structure. MM45T.Li/TK cells were highly sensitive to CCV, ie low concentration of GCV (1 mg/L). MM45T.Li/TK cells can be killed for 3 days. The bystander effect shows mixing 25% MM45T.Li/TK cells with 75% MM45T.Li. Together, more than 90% of cells were found to be killed. Conclusion: Mouse liver cancer cells are highly sensitive to HSV-tk/CCV system and have a significant bystander effect.