The Construction Of The Reversible Immortalized Human Adult Liver Cell RSSR#69/HL-7702 And The Detection Of Its Biological Characteristic

As well as the high incidence and mortality, the liver failure has no effective curable way at present. The animal experiment and the clinical research from home and abroad have proved that the BALSS using the porcine and tumor cells could temporarily and partly substitute the liver to product the liver function. Howere, the animal cells might cause the immumoreaction, zoomosis and ethnics problems; the tumor cells might produce immature function and oncogenensis. For these reasons, the healthy adult liver cell should be an ideal cell for the BALSS. But the fetal liver cell product the immature funcition as well as the ethnics problem. The adult liver cells have limited proliferation in vitro, so it is difficult to cultivate a large quantity of liver cells for the clinical artificial therapy. So we tried to construct an immortalized cells in order to solve the problem.The simian virus 40 was found in the simian kidney cell at 1960s. The simian virus 40 has the function as adjusting the control point of the cell cycle and urging the cell immotalizing. It has been used widely in many kinds of mammalian cells, eg. bone marrow, prostate cells etc. But the immotalized cell has the danger of oncogenesis. The recent research has found that after introducing the HSV-tk gene inside, the DNA synthhesis inside the cell could be cutoff because of the ganciclovir toxicity. There was some reserch indicating that after being transfected the CREase combinated adenovirus, the simian virus 40 T gene could be removed and cause effection of getting rid of the immortalization.In our research, by using the retrovirus vector, the simian virus 40T immortalized gene and HSV-tk suicide gene were introduced into the human liver cell HL-7702. By using the RT-PCR, western blot and indirect immunostain, the exist and expression of the simian virus 40T immortalized gene in the immortalized cell was detected on the gene and protein levels. The exist of the HSV-tk suicide gene in the immortalized cell was detected by the ganciclovir sensitity experiment. The cell surve and the cell cycle was obtained by the MTT stain and the flow cytometry. The liver function gene was dectected through theRT-PCR. The cyp 450ase protein inside the immortalized was detected by the western blot. The configuration and growth of the immortalized cell was observed under the phase contrast microscope. The ultramicro construction of the immortalized cell was obserbed by the tranmission electron microscope.Results:(1) RT-PCR, western blot and indirect immunostain indicated that the reversible immoratalized human liver cell contains the simian virus 40T immortalized gene; (2) the ganciclovir sensitity experiment indicated that the reversible immoratalized human liver cell contains the HSV-tk suicide gene; (3) RT-PCR indicated that the reversible immoratalized human liver cell contains the liver function gene;(4) The cell surve and the cell cycle indicated that the proliferation livingness of the reversible immoratalized human liver cell was stronger than it of the human liver cell HL-7702; (5) Under the the phase contrast microscope and the tranmission electron microscope, the configuration and ultramicro construction of the immortalized cell have no significant difference from the human liver cell HL-7702; (6) The result of the western blot indicated that before and after the immortalization the two kinds of liver cells expressed the cyp 450ase protein inside; (7) The AFP haven't been detected in the supematent of the culture medium of the immortalized liver cell and the human liver cell.This research has constructed a reversible immortalized human liver cell that contains the he simian virus 40T immortalized gene and HSV-tk suicide gene; the immortalized liver cell has a high proliferation ratio; the configuration and biological characteristic has no significant difference from the human liver cell HL-7702; the AFP hasn't detected in the supematent of the the immortalized liver cell . These indicated that the cell could resolve the problem of exiquity of the liver cells for the BALSS, and provide a more ideal cell. It values further research.