α肿瘤坏死因子（TNFα）基因的分离鉴定及在人肝癌细胞中的表达  

PREPARATION OF TNFα cDNA FROM TUMOR INFILTRATING MONONUCLEAR CELLS OF PATIENTS WITH HEPATOCELLULAR CARCINOMA(HCC) FOR THE GENE THERAPY OF HCC

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

机构地区：[1]上海医科大学肝癌研究所,中国科学院上海生物化学研究所

出　　处：《上海医科大学学报》1996年第2期107-110,共4页Journal of Fudan University(Medical Science)

基　　金：中华医学会93583肝癌项目;中国8631021605基金

摘　　要：应用不连续密度梯度法分离获得人肝癌浸润性单个核细胞（ＴＩＭ），ＲＮＡ斑点杂交提示ＴＩＭ中有明显的ＴＮＦαｍＲＮＡ转录。经ＲＴ－ＰＣＲ扩增反应分离得到一特异的７００ｂｐ左右的ＤＮＡ片段，ＤＮＡ序列分析证实这一ＤＮＡ片段中包含编码人ＴＮＦα成熟肽所需的的全部ｃＤＮＡ序列。将这一人ＴＮＦαｃＤＮＡ克隆到真核细胞表达质粒ｐＳＶＫ３，获得真核细胞表达重组ｐＳＶＫ３－ｔｎｆ，用磷酸钙沉淀法将ｐＳＶＫ３－ｔｎｆ导入人肝癌细胞ＳＭＭＣ７７２１中的细胞培养上清中可检测到ＴＮＦα的一过性表达。结果提示ＴＩＭ可成为获取ＴＮＦα基因的来源，获得的ｐＳＶＫ３－ｔｎｆ重组质粒为肝癌的基因治疗奠定了基础。PURPOSE To prepare tumor necrosis factor alpha(TNFα) gene from tumor infiltration mononuclear cells(TIM) and investigate whether TNFα cDNA could be effectively transduced and expressed in SMMC7721 cells,one relatively TNFα-sensitive hepatocellular carcinoma(HCC) cell line.METHODS TIM was obtained from fresh surgical specimen of patients with HCC by Ficoll-Hypaque discontinue gradient method.TNFα mRNA was detected in freshly isoated TIM using dot blot.TNFα complementary DNA(cDNA),prepared with reverse transcript polymerise reaction(RT-PCR) method from the freshly obtained TIM, was confirmed by DNA sequence analysis.TNFα cDNA was transferred into SMM C7721 cells by calcium phosphatemediated transfection method.RESULTS TNFαcDNA was prepared from freshly obtained TIM by RT-PCR method.A recombinant eukaryotic expression vector,pSVK3-tnf,was constructed in which TNFα gene expression was controlled by SV40 promoter and TNFα cDNA was successfully transferred into SMMC7721 cells.CONCLUSIONS The results suggested that TIM could be a new source of preparing TNFα cDNA and pSVK3-tnf might be useful in gene therapy of HCC.

关 键 词：肿瘤坏死因子 肝肿瘤 肿瘤浸润细胞 基因治疗 

分 类 号：R735.705[医药卫生—肿瘤]