The hyposensitization of oval cell to antiproliferative effect of transforming growth factor β1 by hepatitis B virus X gene / 中华外科杂志

<p><b>OBJECTIVE</b>To determine whether hepatitis B virus X (HBX) protein expression affect the oval cells' response to anti-proliferative effect of transforming growth factor β1 (TGFβ1) in oval cells.</p><p><b>METHODS</b>Real-time PCR, Western blot analysis were performed to detect the expression of TGFβRII in HBX-transfected oval cells named HBX-EGFP-LE/6, and EGFP-LE/6, LE/6 control cells. In addition, exogenous TGFβ1 was added into all three oval cell lines, MTT assay was preformed to clarify different responses to the anti-proliferative effect of TGFβ1.</p><p><b>RESULTS</b>The TGFβRII mRNA levels in LE/6 and EGFP-LE/6 cells were (10.2 ± 1.8) and (8.8 ± 0.9) folds of those in HBX-EGFP-LE/6 cells, the difference was significant (P < 0.05). HBX protein expression also reduced the protein levels of TGFβRII in HBX-EGFP-LE/6 oval cells, compared to the control cells. The MTT results exhibited that, after TGFβ1 addition, proliferative inhibition rate in the HBX-EGFP-LE/6 cells was 18.1% ± 1.5% while those in control cells were 42.2% ± 2.8% and 41.9% ± 5.0%, the difference was significant (P < 0.01).</p><p><b>CONCLUSION</b>HBX protein expression affects TGFβRII transcriptional activity and protein synthesis, and insensitive oval cells to anti-proliferative effect of TGFβ1.</p>

Construction of pEGFP-HBx gene recombinant plasmid and establishment of stable transfected HBx gene liver oval cell strain / 中华外科杂志

<p><b>OBJECTIVE</b>To construct pEGFP-HBx eukaryotic expression plasmid and establish stable and effective transfected rat oval cell (LE/6) strain expressing EGFP-HBx fusion protein to explore the roles of HBx gene and oval cell in carcinogenesis of hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>HBx gene with EcoRI and Hind III endo-enzyme sites was obtained by using PCR from plasmid pcDNA3.1-HBx. The purified HBx gene fragment was inserted into pEGFP-N1 expression vector, and the recombinant plasmid pEGFP-HBx was identified by restriction endonuclease and DNA sequencing analysis. LE/6 cells were transfected with recombinant pEGFP-HBx by lipofectamine reagent. Resistant to G418 clones were selected, and expression of EGFP-HBx fusion protein in clones were examined directly with fluorescence microscope, and these clones were isolated and proliferated. The expression of HBx was detected by RT-PCR analysis and immunocytochemistry.</p><p><b>RESULTS</b>Plasmid pEGFP-HBx has whole HBx gene base and correct reading frame as indicated by restriction endonuclease and DNA sequencing analysis. After transfecting with pEGFP-HBx plasmid, LE/6 cell clones expressing EGFP-HBx fusion protein were obtained. RT-PCR analysis and immunocytochemistry showed that HBx gene was only expression in transfected pEGFP-HBx cells.</p><p><b>CONCLUSIONS</b>The pEGFP-HBx recombinant expression vector was successfully constructed, and the stable transfected LE/6 strain expressing EGFP-HBx fusion protein was successfully established. It will be helpful in the further study on the roles of HBx and liver oval cell in carcinogenesis of HCC.</p>