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

来源 :世界胃肠病学杂志(英文版) | 被引量 : 0次 | 上传用户:xiaoxie20092009
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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/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 semiquantitative 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.
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