论文部分内容阅读
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。
Objective To establish a hIL-2 gene-modified human hepatocyte cell line and investigate its effect on its biological activity. Methods Recombinant retrovirus vector pLNCIL-2 was used to transduce hIL-2 gene into human hepatoma cell line L-02. The morphology and cloning rate of hIL-2 gene were detected. The content of hIL-2 in cell culture supernatant was detected, PCR amplification of NeoR gene fragment from cell genomic DNA. Results The proliferation rate and colony formation rate of L-02 / IL-2 cells were lower than those of L-02 / Neo cells and L-02 cells. The content of hIL-2 in the culture medium of L-02 / IL-2 cells reached 32 000 pg / 106 cells · d, and the expression of hIL-2 persisted for more than 10 wk. The NeoR gene fragment (790 bp) was amplified from the genomic DNA of L-02 / IL-2 cells and L-02 / Neo cells. Conclusion The L-02 / IL-2 cell line has been successfully established by recombinant retroviral vector, which lays the foundation for further animal experiments of hepatocyte transplantation.