Transfection and expression of hepatitis B virus x gene and its effect on apoptosis in HL-7702 cells

AIM:To investigate the effects of hepatitis B virus×geneand its protein product HB×Ag on apoptosis in hepatocyteline HL-7702.METHODS:The reconstituted plasmid pcDNA3-x wasestablished through recombination DNA technique;pcDNA3-X was transfected into HL-7702 cells by lipid-mediatedtrasfection.Positive clones were screened by G418,and HL-7702/HBx cells were analysed by the RT-PCR to confirm thesteady expression of X gene in HL-7702 cells.The apoptosisrate in HL-7702 cells was determined by flow cytometry,TUNEL technology,electronic microscope.At the mean time,pcDNA3-X was transfected transiently into HL-7702 cells,and total RNA from HL-7702 cells was extracted 24,48,72,96 and 120 h after the transient transfection,and semi-quantitative analysis was performed by RT-PCR to detectthe expression of HBV X gene.Furthermore,apoptosis ratein HL-7702 cells was determined by flow cytometry analysisat the different times.RESULTS:RT-PCR analysis showed that HBV X gene couldbe expressed stably in HL-7702 cells.Both flow cytometryand TUNEL technology revealed that the apoptosis rates ofHL-7702/HBx cells were much higher than those of HL-7702/pcDNA3 and HL-7702 cells.Furthermore,the apoptoticphenomena and apoptotic body were observed in HL-7702/HBx cells under electronic microscope,but not in HL-7702/pcDNA3 and HL-7702 cells.In the experiment of transienttransfection,RT-PCR reveals that X gene was expressed mostat 72 h after transfection;and the apoptosis rate reachedthe highest at the same time.After that,the apoptosis ratewas reduced with the decrease of the X gene expression.CONCLUSION:HBV X gene and X protein can promote theapoptosis in hepatocyte.And there exist a quantity-effectrelationship between the X gene expression and apoptosisrate in hepatocyte.