脂质体介导pBLAST2-hHGF质粒转染Lewis大鼠肝卵圆细胞

Liposome-mediated pBLAST2-hHGF plasmid transfection in Lewis rat hepatic oval cells

背景:在体外将外源基因导入肝细胞相当困难,而利用肝脏干细胞导入外源性基因较为容易。目前大鼠肝卵圆细胞增殖模型的建立已较为成熟,但作为原代细胞,肝卵圆细胞的稳定培养和传代是较为困难的。目的:建立pBLAST2-hHGF质粒稳定转染大鼠肝卵圆细胞的细胞株,观察转染细胞的生物学特性。方法:取稳定培养的第4代雄性Lewis大鼠肝卵圆细胞,采用脂质体介导DNA转染的方法将pBLAST2-hHGF质粒转染到肝卵圆细胞,然后在倒置显微镜下观察转染细胞的形态变化、细胞增殖情况,检测细胞转染后的增殖分化能力,确定转染细胞的稳定培养条件,使用western blot和ELISA方法检测转染细胞hHGF蛋白的表达。结果与结论:第4代以脂质体介导成功转染pBLAST2-hHGF质粒的肝卵圆细胞在培养基中添加不同剂量和种类细胞因子的条件下转染细胞可稳定传代14代,其形态与未转染细胞比较无明显变化,但生长增殖速度明显快于未转染细胞,去除培养液中的生长因子后pBLAST2-Hhgf/肝卵圆细胞迅速分化为肝细胞和胆管上皮细胞,western blot方法检测转染细胞有hHGF蛋白表达,ELISA法检测培养液中hHGF蛋白含量高。结果提示,使用脂质体介导pBLAST2-hHGF质粒转染肝卵圆细胞方法可靠,转染细胞稳定培养并传代次数长于肝卵圆细胞,转染细胞具备分泌hHGF的能力,可用于后续研究。

BACKGROUND：It is difficult to transfect exogenous gene into hepatocyte in vitro,while that is easily done in liver stem cells.The establishment of rat hepatic oval cells（HOCs） proliferation model is relatively stable at present,but stable culture and passage on primary HOCs are difficult.OBJECTIVE：To establish pBLAST2-hHGF plasmid stably transfected cell line in rat hepatic oval cells,and observe the biological characteristics of transfected cells.METHODS：Liposome-mediated gene transfection was used to transfer pBLAST2-hHGF plasmid into the fourth generation of hepatic oval cells derived from male Lewis rats.Morphological changes and cell proliferation of transfected cells were observed under inverted phase contrast microscope.Proliferation and differentiation capability of transfected cells were measured and stable culture conditions were explored.Western blot assay and enzyme linked immunosorbent assay were used to test human hepatocyte growth factor（hHGF） protein expression on transfected cells.RESULTS AND CONCLUSION：The fourth generation pBLAST2-hHGF/HOCs could be passaged steadily to 14 generations without significant morphological changes under the culture conditions containing various doses and types of cytokines.The growth and proliferation speed of transfected cells were obviously faster than that of HOCs.After removing growth factors mentioned above,pBLAST2-hHGF/HOCs quickly differentiated into hepatocytes and biliary epithelial cells in vitro.hHGF protein could be tested in pBLAST2-hHGF/HOCs using Western blot assay and it also could be tested with high level in culture fluid using enzyme linked immunosorbent assay.Results indicate that liposome-mediated gene transfection in this experiment was reliable and the stable passage time of pBLAST2-hHGF/HOC was longer than that of HOCs.pBLAST2-hHGF/HOCs had the ability to secrete hHGF protein and this cell line could be used in following experiment.