The Induction Of HBX On The Apoptosis Of Different Hepatocytes

Objective:Construct the plasmid of PEGFP-N1-HBX.To study the effect of NF-κB signaling pathway for HBX on the apoptosis of different hepatocyte.Methods:1.Cells LO2and HepG2were cultured in vitro cell culture.2.We transfected LO2and HepG2with the plasmid of PEGFP-N1-HBX.The expression of EGFP was examined with fluorescence microscope observations.Western Blot were facilitated to observe the expression of HBX protein.3.NF-κB signaling pathway blocker PDTC was introduced to cut off its signal transduction.4.Flow cytometry were applied to study the cell cycle and apoptosis of different hepatocytes.5. Western Blot were facilitated to observe the expression of NF-κB protein before and after the HBX transfection as well as treating with PDTCResults:1.The specific expression of EGFP gene was only observed in transfected cells,but not in non-transfected cells (Figl)2.The target protein HBX were detected in the transfected LO2and HepG2cells(Fig2).3.The apoptosis of L02/HBX significantly increased compared with control. The proportion of cells in G0/G1stage increased with cells in S and G2/M stage decreased.The apoptosis of HepG2/HBX significantly decreased compared with control. The proportion of cells in G0/G1stage decreased with cells in S and G2/M stage increased.After PDTC treatment,The proportion of L02/HBX/PDTC cells increased significantly in G0/G1phase, but decreased remarkably in S and G2/M phase and the apoptosis rate was at a significantly higher level compared with L02/HBX cells.The apoptosis and the proportion of cells in G0/G1phase of HepG2/HBX/PDTC are increased significantly, and decreased remarkably in S and G2/M phase, but the apoptosis rate was still lower than the HepG2cells.4.The expression of NF-κB protein in L02/HBX was significantly decreased and increased in HepG2/HBX cells compared with control.But there were nearly no expression in L02/HBX/PDTC and HepG2/HBX/PDTC cells.Conclusion:1. LO2-HBX cell line and HepG2-HBX cell line stably expressing HBX is established successfully. 2.After stably transfecting HBX gene,the HBX can retard the human normal hepatic cell line cell cycles and facilitate the apoptosis through down-regulate the expression of NF-κB protein.the HBX can accelerate the human hepatocyte cell line cell cycles and suppress the apoptosis through up-regulate the expression of NF-κB protein.