逆转录病毒介导IL－2基因导入肝癌细胞的研究  

Retroviral mediated IL-2 gene transfer into human liver carcinoma cell

作　　者：王学浩[1,2] 孙耀明[1,2] 杜竞辉[1,2] 单祥年 鲁晓暄[1,2] 张晓明 

机构地区：[1]南京医科大学第一附属医院肝胆外科 [2]南京铁道医学院分子生物教研室

出　　处：《肿瘤防治研究》1996年第5期289-291,共3页Cancer Research on Prevention and Treatment

摘　　要：利用逆转录病毒载体系统ＰＡ３１７／ＬＮＣ－ＩＬ－２所产生的含有ＩＬ－２基因及新霉素抗性基因（ｎｅｏｒ）的重组病毒，经共培养转染ＳＭＭＣ－７７２１肝癌细胞株。ＩＬ－２基因受巨细胞病毒早期启动子调控，转染后的细胞置于含新霉素类毒性药物Ｇ４１８（４００ｕｇ／ｍｌ）培养基内培养、筛选３０天，结果果转染率约为１～２％。对存活下来的转染有ＩＬ－２基因和新霉素抗性基因的细胞形态和生长情况进行观察，同时从水平及蛋白质水平对外源性基因的转录和翻译进行检测。结果表明：ＳＭＭＣ－７７２１肝癌细胞在ＩＬ－２基因转染前后形态及生长情况无明显变化；培养上清活性检测发现转染后为转染前的２０～３０倍；逆转录ＰＣＲ分析显示转染后的ＩＬ－２ｍＲＮＡ量明显高于转染前。研究表明我们已成功地将ＩＬ－２基因导入肝癌细胞并获得持续、稳定、高效表达。Human liver carcinoma cell line SMMC-7721 were transfected by replication deficient retroviral vectr produced by PA317/LNC-IL-2 packaging cell line containing the interleukin-2 gene and the gene coding for resistance to neomycin (neor).This process was carried out through cocultivation. IL-2 gene was under the control of the cytomegalovirus (CMV) early promoter. After transfection the tumourcells were cultured and selected for thirty days in G418 culture medium containing G418 (400μg/ml)-a neomycin analogue toxic to eukaryotic cells. Cells which integrated with neomycin resistance gene and human interleukin-2 gene were survival.The transfection rate was 1￣2%. Growth and shape of the genetically modified SMMC-7721 cells were observed.Successful gene insertion was confirmed by determining the transcription and translation of exogenous interleukin-2 gene. The result showed that the shape and growth of 7721-transduced cells had no obvious variation as compared with those of the contrlo;the biologic activity of IL-2 in the culture supernatand increased to 20￣30folds more than those of the control; reverse transcriptions polymerase chain reaction (RT-PCR) showed hIL-2mRNA was obviously elevated. The study indicated that we have transferred successfully IL-2gene into human liver carcinoma cell line and obtained continuous, stable,efficient expression.

关 键 词：逆转录病毒载体 白细胞介素 肝肿瘤 免疫疗法 

分 类 号：R735.705[医药卫生—肿瘤]