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 x gene and its protein product HBxAg on apoptosis in hepatocyte line HL-7702. METHODS: The reconstituted plasmid pcDNA3-x was established through recombination DNA technique; pcDNA3X was transfected into HL-7702 cells by lipid-mediated trasfection. Positive clones were screened by G418, and HL7702/HI3x cells were analysed by the RT-PCR to confirm the steady expression of X gene in HL-7702 cells. The apoptosis rate 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 semiquantitative analysis was performed by RT-PCR to detect the expression of HI3V X gene. Furthermore, apoptosis rate in HL-7702 cells was determined by flow cytometry analysis at the different times. RESULTS: RT-PCR analysis showed that HI3V X gene could be expressed stably in HL-7702 cells. Both flow cytometry and TUNEL technology revealed that the apoptosis rates of HL-7702/HI3x cells were much higher than those of HL-7702/ pcDNA3 and HL-7702 cells. Furthermore, the apoptotic phenomena and apoptotic body were observed in HL-7702/ HI3x cells under electronic microscope, but not in HL-7702/ pcDNA3 and HL-7702 cells. In the experiment of transient transfection, RT-PCR reveals that X gene was expressed most at 72 h after transfection; and the apoptosis rate reached the highest at the same time. After that, the apoptosis rate was reduced with the decrease of the X gene expression. CONCLUSION: HBV X gene and X protein can promote the apoptosis in hepatocyte. And there exist a quantity-effect relationship between the X gene expression and apoptosis rate in hepatocyte.

AIM:To investigate the effects of hepatitis B virus×gene and its protein product HB×Ag on apoptosis in hepatocyte line HL-7702. METHODS:The reconstituted plasmid pcDNA3-x was established through recombination DNA technique;pcDNA3- X was transfected into HL-7702 cells by lipid-mediated trasfection.Positive clones were screened by G418,and HL- 7702/HBx cells were analysed by the RT-PCR to confirm the steady expression of X gene in HL-7702 cells.The apoptosis rate 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 detect the expression of HBV X gene.Furthermore,apoptosis rate in HL-7702 cells was determined by flow cytometry analysis at the different times. RESULTS:RT-PCR analysis showed that HBV X gene could be expressed stably in HL-7702 cells.Both flow cytometry and TUNEL technology revealed that the apoptosis rates of HL-7702/HBx cells were much higher than those of HL-7702/ pcDNA3 and HL-7702 cells.Furthermore,the apoptotic phenomena 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 transient transfection,RT-PCR reveals that X gene was expressed most at 72 h after transfection;and the apoptosis rate reached the highest at the same time.After that,the apoptosis rate was reduced with the decrease of the X gene expression. CONCLUSION:HBV X gene and X protein can promote the apoptosis in hepatocyte.And there exist a quantity-effect relationship between the X gene expression and apoptosis rate in hepatocyte.