增强型绿色荧光蛋白基因标记结肠癌LoVo细胞的实验研究

Gene marking of human colon cancer LoVo cells with enhanced green fluorescent protein

目的 建立增强型绿色荧光蛋白 (EGFP)基因标记的结肠癌细胞系。方法 构建含EGFP单基因的逆转录病毒载体G1FP ,用脂质体转染结合交互感染的方法获得高滴度的双嗜型EGFP病毒 ,并转导人结肠癌细胞系LoVo;EGFP基因的转移与表达分别以聚合酶链反应 (PCR)和流式细胞术 (FCM)分析。结果 将G1FP载体转染的GP +E 86和GP +envAm12细胞共培养 2周后 ,取单嗜型EGFP病毒感染双嗜型包装细胞获得病毒生产细胞Am12 /G1FP ,所有细胞均表达高水平的EGFP ,其滴度为 1.1× 10 5感染性颗粒 /ml。双嗜型EGFP转导的LoVo细胞可稳定发出明亮的绿色荧光信号。PCR分析显示 ,LoVo/GFP细胞内整合有EGFP原病毒。结论 LoVo/GFP细胞可长期表达荧光 ,可为体内研究结肠癌微转移提供有效模型。

Objective The aim of this study is to obtain a human colon cancer cell line marked with enhanced green fluorescent protein (EGFP) gene.Methods The G1FP retroviral vector, carrying EGFP gene, was constructed. When combined with liposome mediated transfection with cross infection, EGFP virus producer cells were generated. The human colon cancer LoVo cells were transduced with EGFP retrovirus. The integration and expression of EGFP gene were analyzed with polymerase chain reaction (PCR) and flow cytometry.Results The ecotropic EGFP virus was obtained by cocultivation of packging cells for two weeks, while the amphotropic virus producer cells Am12/G1FP, expressing high level EGFP, were generated through cross infection with a titer of 1.1×10 5 infectious particles/ml. The genetically modified LoVo/GFP cells gave off strikingly bright green fluorescence, in which the integration of EGFP provirus was confirmed by PCR analysis.Conclusion The LoVo/GFP cells, which express high level green fluorescence, may provide an useful tool for the study of micrometastasis of colon cancer in vivo. [