AIM: To investigate whether the formation of aggregated HBx has a potential linking with its cellular responses.METHODS: Recombinant HBx was expressed in Escherichia coli and purified by Ni-NTA metal-affinity chromato...AIM: To investigate whether the formation of aggregated HBx has a potential linking with its cellular responses.METHODS: Recombinant HBx was expressed in Escherichia coli and purified by Ni-NTA metal-affinity chromatography.Anti-HBx monoclonal antibody was developed for immunocytochemical detection. Bicistronic expression vector harboring full-length DNA of HBx was employed for transfection of human HepG2 ceils. Immunocytochemical staining was used to examine the intracellular HBx aggregates in cells. The effects of HBx aggregation on cell cycle and apoptosis were assessed by flow cytometry.RESULTS: Immunocytochemical staining revealed most of the HBx was formed intracellular aggregate in cytoplasm and frequently accumulated in large granules. Flow cytometry analysis showed that HepG2 cells transfected with vector harboring HBx significantly increased apoptosis and largely accumulated in the G0-G1 phase by maintenance in serum medium for 36 hours. Control cells without HBx aggregates in the presence of serum entered S phase and proliferated more rapidly at the same time.EGFP fluorescence in HBx expression cells was significantly decreased.CONCLUSION: Our observations show that cells with HBx aggregate undergo growth arrest and apoptosis, whereas control cells without HBx remain in growth and progression into S phase. Our data may provide helpful information to understand the biological effects of HBx aggregates on cells.展开更多
文摘AIM: To investigate whether the formation of aggregated HBx has a potential linking with its cellular responses.METHODS: Recombinant HBx was expressed in Escherichia coli and purified by Ni-NTA metal-affinity chromatography.Anti-HBx monoclonal antibody was developed for immunocytochemical detection. Bicistronic expression vector harboring full-length DNA of HBx was employed for transfection of human HepG2 ceils. Immunocytochemical staining was used to examine the intracellular HBx aggregates in cells. The effects of HBx aggregation on cell cycle and apoptosis were assessed by flow cytometry.RESULTS: Immunocytochemical staining revealed most of the HBx was formed intracellular aggregate in cytoplasm and frequently accumulated in large granules. Flow cytometry analysis showed that HepG2 cells transfected with vector harboring HBx significantly increased apoptosis and largely accumulated in the G0-G1 phase by maintenance in serum medium for 36 hours. Control cells without HBx aggregates in the presence of serum entered S phase and proliferated more rapidly at the same time.EGFP fluorescence in HBx expression cells was significantly decreased.CONCLUSION: Our observations show that cells with HBx aggregate undergo growth arrest and apoptosis, whereas control cells without HBx remain in growth and progression into S phase. Our data may provide helpful information to understand the biological effects of HBx aggregates on cells.
