Stable Expression Of HBx Gene And The Influence Of HBx Gene To The Proliferation And Apoptosis Of L0₂ Cells

Objective Clone HBx gene to Human liver cells L0₂ using retroviral vector. L0₂ cells expressing HBx protein stably provide a cell model to study the relationship between HBx protein and biological behavior of hepatocellular carcinoma.Investigate on the effect of HBx protein to the the L0₂ cell proliferation, cell cycle and the influence HBx protein to the the L0₂ in serum-free medium., and settle foundation for the further study to discuss its possible molecular mechanism of HBx protein to promoting the apoptosis and the formation of HBx protein in HCC.Methods HBx gene was obtained through PCR technology. After digestion with Salâ… and Bglâ…¡, the fragment of X and pSEB-Flag were connected to establish reconstituted plasmid pSEB-Flag-HBx.We transfected pSEB-Flag-HBx into 293 cells, collected retrovirus, and infected L0₂ cells. After selected with Blasticidin, the mRNA and protein steady expression of HBx were determined by the reverse transcription PCR and Western blot respectively. MTT assay was used to analyze the effect of HBx protein on L0₂ cell proliferation; flow cytometry was used to investigate the influence of HBx protein on the L0₂ cell cycle. L0₂-HBx cells were cultured without serum. After serum-free cultivation, the cell proliferation was assessed by MTT assay and flow cytometry was used to investigate the influence of HBx protein on the L0₂ cell cycle. Hoechst 33258 stainings were used to investigate the influence of cell apoptosis of HBx protein without serum.Results HBx full-length gene was obtained by PCR technology from pAd-track-HBx plasmid. The fragment of HBx and pSEB-Flag were connected to establish reconstituted plasmid pSEB-Flag-HBx. Reconstituted retroviral plasmid was amplified by PCR and digested with Salâ… and Bglâ…¡, and there was a band about 500bp after a 1% agarose gel electrophoresis, showing HBx gene was successfully cloned to the plasmid. Retrovirus infected L0₂ cells, selecting with Blasticidin, and we got resistant cells, which demonstrated that the retroviral plasmid DNA had been transfected into L0₂ cells successfully. The HBx mRNA and protein steady expression had been determined by RT-PCR and Western blot respectively. There was no significant difference on proliferation detected by MTT between L0₂-HBx cells and L0₂-Rv cells; and no significant difference on cell cycle was detected by flow cytometry between L0₂-HBx cells and L0₂-Rv cells. After serum-free cultivation, the cell proliferation was no significant difference detected by MTT assay and L0₂-HBx cells were arrested in G1 phase more obviously. L0₂-HBx cells has more apoptosis detected by Hoechst 33258 stainings.Conclusion 1. L0₂-HBx cells, expressing HBx protein steadily, was successfully established , which was an ideal model to study the effect of HBx on the development of hepatocelular carcinoma. 2. HBx protein had no effect on cell proliferation. 3. HBx protein had no effect on cell cycle. 4. Without serum, HBx protein had no effect on cell proliferation. 5. HBx protein could reinforce cell cycle G1 phase arrest. 6. Serum-free processing and HBx protein could promote cell apoptosis.