Influence On Biological Character Istics Of Hepatocytes Transfected With HTERT Gene By Lentivirus

AIM:To investigate the influence on the biological characteristics of human hepatic cell line chang liver transfected with h-TERT gene mediated by lentiviral vector.METHODS:The positive clones are screened by blasticidin in human hepatic cell line chang liver transfected with h-TERT gene mediated by lentiviral vector. Real-time fluorescence polymerase chain reaction method is used for detection of liver transfected chang liver cells h-TERT-mRNA gene expression level.The method of TRAP-PCR and ELISA are used for telomerase activity before and after transfection in chang liver hepatocytes. Morphology and growth proliferation of liver cells before and after transfection can be observed by inverted phase contrast microscope. The changes of cell growth and cell cycle before and after transfection can observed by MTT and flow cytometry, respectively. The content and mRNA expression of albumin in supernatant before and after transfection can observed by biochemical analyzer and real-time fluorescence polymerase chain reaction, respectively. The expressions of glycogen before and after transfection were identified by periodic acid-schiff.RESULTS:Transfected liver cells show h-TERT gene redundant expression and telomerase activity has increased. H-TERT-chang liver shows oval, spindle or polygonal, epithelial cells adherent growth, which is without significant difference compared with cells before transfected. Cell growth curves show that h-TERT-chang liver has faster growth spead than change liver. By observing the cell cycle further,we found that Gl phase cells decrease, G2/M phase and S phase cells increase and PI increase which suggest that h-TERT-chang liver proliferation ability is stronger than chang liver. Biochemical results showed that the supernatant of transfected liver cells still maintain the ability of the synthesis and secretion of albumin. The periodic acid-schiff staining before and after transfection results show that liver cells transfected can still keep the ability of glycogen synthesis.CONCLUSIONS:H-TERT-chang liver cells with hTERT gene by lentivirus can keep intrinsic morphology, remarkable extended survival time, stronger proliferation ability. These cells have functions of secreting albumin and synthesizing glycogen. Above results may provide a kind of cheaper, easier available and safer hepatocyte source for clinical application on creaturely factitial liver and set up a new experimental basis and biological model for hepatocyte replantation and drugs screen in vitro.