Chen Hong-Ying, Tang Nan-Hong, Li Xiu-Jin, Zhang Sheng-Jun, Chen Zhi-Xin, Wang Xiao-Zhong
Department of Gastroenterology, Union Hospital, Fujian Medical University, Fuzhou 350001, Fujian Province, China.
World J Gastroenterol. 2004 Apr 1;10(7):959-64. doi: 10.3748/wjg.v10.i7.959.
To investigate the effects of hepatitis B virus x gene and its protein product HBxAg on apoptosis in hepatocyte line HL-7702.
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.
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.
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.
研究乙型肝炎病毒X基因及其蛋白产物HBxAg对肝细胞系HL-7702细胞凋亡的影响。
采用重组DNA技术构建重组质粒pcDNA3-x；通过脂质介导转染法将pcDNA3-X转染至HL-7702细胞。用G418筛选阳性克隆，采用RT-PCR分析HL-7702/HBx细胞，以确认X基因在HL-7702细胞中的稳定表达。采用流式细胞术、TUNEL技术、电子显微镜检测HL-7702细胞的凋亡率。同时，将pcDNA3-X瞬时转染至HL-7702细胞，在瞬时转染后24、48、72、96和120小时提取HL-7702细胞的总RNA，采用RT-PCR进行半定量分析以检测HBV X基因的表达。此外，在不同时间通过流式细胞术分析测定HL-7702细胞的凋亡率。
RT-PCR分析显示HBV X基因可在HL-7702细胞中稳定表达。流式细胞术和TUNEL技术均显示HL-7702/HBx细胞的凋亡率远高于HL-7702/pcDNA3细胞和HL-7702细胞。此外，在电子显微镜下观察到HL-7702/HBx细胞中有凋亡现象和凋亡小体，而HL-7702/pcDNA3细胞和HL-7702细胞中未观察到。在瞬时转染实验中，RT-PCR显示X基因在转染后72小时表达量最高；同时凋亡率也达到最高。此后，随着X基因表达量的降低，凋亡率也随之降低。
HBV X基因和X蛋白可促进肝细胞凋亡。并且X基因表达与肝细胞凋亡率之间存在量效关系。
