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采用比利时Gent大学的质粒PATHNF(内含人TNF-α-cDNA),以及英国伦敦大学的重组逆转录病毒载体pRUFneo.将人TNF基因克隆到重组逆转录病毒载体pRUFneo,构建了含人TNFL-a基因重组逆转录病毒载体RUF-TNF.将RUF-TNF转染包装细胞FlyA13,经G41筛选获得稳定分泌含人TNF基因重组逆转录病毒的细胞株FlyRUFN14.FlyRUFN14产生的病毒上清液能直接转染靶细胞Saos-2.采用这一基因转移系统,将TNF基因转染到两株人肝癌细胞株SMMG-7721及BEL-7404,获得转基因肝癌细胞SMMC-7721 TNF及BEL-7404TNF.TNF基因对人肝癌细胞生长的影响采用细胞学实验及裸鼠实验两种方法进行研究.①细胞学实验:将转基因肝癌细胞SMMC-7721TNF及BEL-7404TNF作为实验组,原来的未转基因肝癌细胞株SMMC-7721及BEL-7404作为对照组,用MTT法研究两组的生长速度.结果:转基因肝癌细胞SMMG-7721TNF及BEL-7404TNF的生长速度明显慢于未转基因肝癌细胞,差别有显著意义(P<0.01).②裸鼠实验:
University of Gent, Belgium, using plasmid PATHNF (containing human TNF-α-cDNA), and recombinant retroviral vector pRUFneo University of London. The recombinant human TNF gene was cloned into retroviral vector pRUFneo, plasmids containing human TNFL-a recombinant retroviral vector RUF-TNF. the RUF-TNF transfected packaging cells FlyA13, G41 screened by stable cell lines containing the secreted recombinant human TNF FlyRUFN14.FlyRUFN14 retrovirus produced viral supernatant can be directly transfected Target cell Saos-2. Using this gene transfer system, the TNF gene was transfected into two human hepatocellular carcinoma cell lines SMMG-7721 and BEL-7404. Transgenic hepatoma cells SMMC-7721 TNF and BEL-7404 TNF. TNF gene pairs were obtained. Effect hepatoma cell using cytological methods in nude mice experiment and its two kinds of experimental studies .① cytology: transgenic hepatoma cells SMMC-7721TNF BEL-7404TNF and experimental group, the original non-transgenic SMMC-7721 hepatoma cell line and BEL-7404 was used as a control group. The growth rate of the two groups was studied by MTT assay. Results: The growth rate of SMMG-7721TNF and BEL-7404TNF in transgenic hepatoma cells was significantly slower than that of non-transgenic hepatoma cells. Significance(P<0.01).2 Nude mouse experiment: