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


CONCLUSIONS
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.
Chen
Hong-Ying
HY
Department of Gastroenterology, Union Hospital, Fujian Medical University, Fuzhou 350001, Fujian Province, China. drwangxz@pub6.fz.fj.cn
Tang
Nan-Hong
NH
Li
Xiu-Jin
XJ
Zhang
Sheng-Jun
SJ
Chen
Zhi-Xin
ZX
Wang
Xiao-Zhong
XZ
D017209:Apoptosis / Q000187:drug effects*; D002460:Cell Line; D005434:Flow Cytometry; D015870:Gene Expression; D022781:Hepatocytes / Q000187:drug effects* / Q000502:physiology / Q000648:ultrastructure; D008854:Microscopy, Electron; D015534:Trans-Activators / Q000235:genetics* / Q000494:pharmacology*; D014162:Transfection; D054334:Viral Regulatory and Accessory Proteins
D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein