逆转录病毒介导的人TNF在人肝癌细胞系(SMMC)中的表达  

Expression and antitumor activity of retrovirus-mediated tumor necrosis factor a gene in human hepato cellular carcinoma cells

作　　者：冯学胜[1] 郑仲承[2] 汤钊猷[1] 戈凯[2] 王立群[2] 薛琼[1] 孙兰英[2] 刘新垣[2] 

机构地区：[1]上海医科大学肝癌研究所,200032 [2]中国科学院上海生物化学研究所

出　　处：《中华微生物学和免疫学杂志》1996年第1期33-36,共4页Chinese Journal of Microbiology and Immunology

基　　金：本课题受美国中华医学基金会CMB93583;中国"863"计划8610605项目资助

摘　　要：利用逆转录病毒载体ＬＸＳＮ构建了含人ＴＮＦａ基因的重组逆转录病毒载体Ｌ－ｔｎｆＳＮ。磷酸钙沉淀法将重组载体引入病毒包装细胞ＰＡ３１７，挑选Ｇ４１８抗性克隆，用ＮＩＨ３Ｔ３细胞测定病毒滴度，获得滴度为１×１０５ＣＦＵ／ｍｌ的细胞克隆。应用这一重组病毒感染人肝癌细胞ＳＭＭＣ７７２１，经Ｇ４１８筛选获得抗性克隆。ＰＣＲ可从转导的细胞ＤＮＡ中扩增出ＴＮＦａ基因片段，提示目的基因完整地整合在细胞基因组中。测定转导细胞培养上清中ＴＮＦａ活性，结果显示ＴＮＦａ有相对稳定的表达（１５０～３７０Ｕ／１０６ｃｅｌｌｓ／２４小时）。实验还表明转导细胞的体外生长能力无明显变化，但是其在裸鼠中的致瘤性却明显降低，提示逆转录病毒介导ＴＮＦａ基因对人原发性肝场可能有一定的治疗作用。One recombinant retroviral vector L-tnfSN was constructed with human tumor necrosis factor alpha (TNFa ) cDNA and a retroviral vector LXSN. After identified by restriction endonuclease digestion , L-tnfSN was introduced into packaging cell line PA31 7 by calcium phosphate transfection method. Several G418-resistant colonies were obtainted and the virus titer in supernatant of the colonies were determined by NIH 3T3 cells , the highest of which was about 1 X 105 CFU/ml. The supernatant was then used to infect the human hepatocellular carcinoma (HCC) cell line SMMC7721. After screened by G418 , several G41 8-resistant colonies of HCC cells (named as SMMC7 72 1-TNF ) were established. The biological activity of TNFa in culture - supernatant of SMMC7721-TNF cells was monitored which was in the range of 150-370U/106 cells/24h. The integration of TNFa cDNA was identified by PCR method in the genomic DNA of these colonies. Compared with its parental cell lines , the proliferative ability of SMMC7721-TNF cells did not change. However the tumorigenicity of SMMC7 721-TNF in nude mice decreased greatly that suggests a potential role in treatment of HCC.

关 键 词：逆转录病毒 基因治疗 肿瘤坏死因子 肝癌 

分 类 号：R735.7[医药卫生—肿瘤]