逆转录病毒载体介导IL-2基因转染人肝细胞的建立及其部分生物学性状

目的　　建立ｈＩＬ－２基因修饰的人肝细胞株并探讨对其生物学活性的影响。　方法应用重组逆转录病毒载体ｐＬＮＣＩＬ－２，将ｈＩＬ－２基因导入人肝细胞系Ｌ－０２，观察其形态及克隆形成率的变化，检测细胞培养上清中ｈＩＬ－２的含量，以及进行转染细胞基因组ＤＮＡ　ＮｅｏＲ基因片段的ＰＣＲ扩增。结果Ｌ－０２／ＩＬ－２细胞的增殖速度和克隆形成率低于Ｌ－０２／Ｎｅｏ细胞和Ｌ－０２细胞。Ｌ－０２／ＩＬ－２细胞的培养液中ｈＩＬ－２的含量达３２　０００　ｐｇ／１０６ｃｅｌｌｓ·ｄ，并可持续表达１０　ｗｋ以上。从Ｌ－０２／ＩＬ－２细胞和Ｌ－０２／Ｎｅｏ细胞基因组ＤＮＡ中，均扩增到ＮｅｏＲ基因片段（７９０　ｂｐ）。结论应用重组逆转录病毒载体成功地建立了Ｌ－０２／ＩＬ－２细胞株，为进一步进行肝细胞移植动物实验奠定了基础。

Establishment of human hepatocytes transfected by pLNC-IL-2 and its partial biological activities

Aim To establish human hepatocyte line L-02 transfected with pLNCIL-2 and explore the effect of this vector on the biological feasures of the cells. Methods Human interleukin-2 gene was transferred into the L-02 cells by the aid of recombinant retroviral vector pLNCIL-2, then change of morphology and colonogeneicity of the transferred cells were observed. At the same time, the level of hIL-2 secretion in cultural supernatant was detected and NeoR gene were amplified by PCR. Results Proliferation and colonogeneicity of the L-02 /IL-2 cells were lower than those of L-02/Neo and L-02 cells. hIL-2 level secreted reached to 32 000pg/106cells per day and the secretion could gone on for more than ten weeks. In addition, NeoR gene segment was obtained by PCR from both L-02/IL-2 and L-02/Neo cell's genomic DNA. ConclusionhIL-2 gene has been integrated successfully into genomic DNA of human hepatocytes L-02. This result lay the foundation for further research.