Abstract:

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.