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逆转录病毒介导IL-2基因导入肝癌细胞的研究[J]. 肿瘤防治研究, 1996, 23(5): 289-291.
引用本文: 逆转录病毒介导IL-2基因导入肝癌细胞的研究[J]. 肿瘤防治研究, 1996, 23(5): 289-291.
Retroviral mediated IL-2 gene transfer into human liver carcinoma cell[J]. Cancer Research on Prevention and Treatment, 1996, 23(5): 289-291.
Citation: Retroviral mediated IL-2 gene transfer into human liver carcinoma cell[J]. Cancer Research on Prevention and Treatment, 1996, 23(5): 289-291.

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

  • 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CPR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?

     

    Abstract: 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.

     

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