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目的 :研究肿瘤的自杀基因疗法 ,建立含单纯疱疹病毒胸苷激酶 (HSVtk)基因重组逆转录病毒包装细胞PA317/TK。 方法 :应用脂质体转移方法将逆转录病毒载体GINaTK导入PA317,经G4 18筛选阳性克隆细胞后分别用PCR和扫描电镜检测HSVtk基因整合和病毒颗粒分泌情况。 结果 :逆转录病毒载体GINaTK导入PA317,G4 18后筛选得到阳性克隆细胞 ,命名为PA317/TK。PCR检测PA317/TK细胞出现一特异性 4 0 4bp阳性条带 ,而PA317细胞没有扩增出相应条带。PA317/TK细胞经扫描电镜检测见其表面有颗粒状突起 ,其周围有许多呈团状的病毒颗粒。证实含HSVtk基因重组逆转录病毒存在于包装细胞中。 结论 :成功建立含HSVtk基因重组逆转录病毒包装细胞
OBJECTIVE: To study the tumor suicide gene therapy and to establish the recombinant retrovirus packaging cell line PA317 / TK containing herpes simplex virus thymidine kinase (HSVtk) gene. Methods: The retroviral vector GINaTK was transfected into PA317 by lipofectamine. The positive clones were screened by G418. The integration of HSVtk gene and the secretion of virus particles were detected by PCR and scanning electron microscopy. RESULTS: The retroviral vector GINaTK was transfected into PA317 and G4 18 cells, and then the positive clones were obtained and named as PA317 / TK. PCR detected PA317 / TK cells showed a specific 4 0 4bp positive bands, PA317 cells did not amplify the corresponding band. PA317 / TK cells by scanning electron microscopy showed that there are granular protrusions on its surface, surrounded by many clusters of virus particles. It was confirmed that the recombinant retrovirus containing the HSVtk gene was present in the packaging cells. Conclusion: Recombinant retroviral packaging cells containing HSVtk gene were successfully established