目的： 研究乙型肝炎病毒X蛋白（hepatitis B virus X protein，HBX）通过核转录因子NF-κB信号通路对不同肝细胞系凋亡的诱导作用。 方法： 建立稳定转染PEGFP-N1-HBX质粒的人正常肝细胞系L02（L02/HBX）和人肝癌细胞系HepG2（HepG2/HBX），用特异性NF-κB阻断剂吡咯二硫代氨基甲酸酯（pyrrolidine dithiocarbamate，PDTC）阻断NF-κB信号通路，流式细胞术检测PEGFP-N1-HBX转染前后及加入PDTC 前后L02和HepG2细胞的细胞周期与凋亡，Western blotting检测NF-κB 的表达。 结果： 成功构建了稳定转染PEGFP-N1-HBX的L02/HBX和HepG2/HBX细胞。与对照组L02细胞相比，L02/HBX细胞凋亡率明显增加\[(31.31±0.51)% vs (14.05±0.09)%，P<0.05\]；其G0/G1期细胞比例显著增加，S期和G2/M期细胞比例减少。与对照组HepG2细胞相比，HepG2/HBX细胞凋亡率显著降低\[(1.21±0.04)% vs (10.26±0.10)%，P<0.05\]；其G0/G1期细胞比例显著减少，S期和G2/M期细胞比例增加。PDTC作用后，L02/HBX/PDTC组凋亡率\[(40.33±0.07)%\]及G0/G1 期细胞比例较L02/HBX组显著增加，G2/M期细胞比例明显减少，而HepG2/HBX/PDTC组凋亡率\[(5.45±0.07)%\]及G0/G1 期细胞比例较HepG2/HBX组显著增加，S期和G2/M期细胞比例明显减少，但其凋亡率仍低于对照HepG2细胞。Western blotting结果显示，L02/HBX细胞NF-κB表达显著下调，而HepG2/HBX细胞NF-κB表达显著增加，L02/HBX/PDTC和HepG2/HBX/PDTC细胞NF-κB几乎不表达。 结论： HBX可下调正常肝细胞L02 NF-κB蛋白的表达，阻滞细胞周期，促进细胞凋亡；而HBX可增加肝癌细胞系HepG2中NF-κB蛋白的表达，加速细胞周期，抑制肝癌细胞凋亡。

Objective:To study the effect of NF-κB signaling pathway for hepatitis B virus X protein (HBX) on the apoptosis of different hepatocyte lines. Methods: To establish the normal hepatic cell LO2 and hepatic cancer cell HepG2 stably transfected with PEGFP-N1-HBX plasmid (L02/HBX or HepG2/HBX cells). NF-κB signaling pathway inhibitor pyrrolidine dithiocarbamate (PDTC) was used to cut off NF-κB signal transduction in LO2 and HepG2 cells. Flow cytometry was applied to study the cell cycle and apoptosis of LO2 and HepG2 cells before and after PEGFP-N1-HBX transfection as well as before and after PDTC treatment. Western blotting was used to examine the expression of NF-κB. Results: LO2/HBX cells and HepG2/HBX cells stably transfected with PEGFP-N1-HBX were established successfully. The apoptosis of L02/HBX cells significantly increased compared with the control L02 cells \[(31.31±0.51)% vs (14.05±009)%, P<0.05\], and the proportion of cells in G0/G1 stage increased with cells in S and G2/M stage decreased. The apoptosis of HepG2/HBX cells significantly decreased compared with the control HepG2 cells (\[1.21±0.04\]% vs \[1026±0.10\]%, P<0.05), and the proportion of cells in G0/G1 stage decreased with cells in S and G2/M stage increased. After PDTC treatment, the proportion of L02/HBX/PDTC cells in G0/G1 phase increased significantly (\[40.33±0.07\]%), while that in S and G2/M phase decreased remarkably, and the apoptosis rate (\[5.45±007\]%) was at a significantly higher level compared with L02/HBX cells. The apoptosis and the proportion of cells in G0/G1 phase of HepG2/HBX/PDTC were increased significantly, and decreased remarkably in S and G2/M phase, while the apoptosis rate was still lower than the HepG2 cells. The expression of NF-κB protein was significantly decreased in L02/HBX cells but increased in HepG2/HBX cells compared with the control cells. There was almost no expression of NF-κB protein in L02/HBX/PDTC and HepG2/HBX/PDTC cells. Conclusion: HBX can retard the cell cycle of normal hepatic L02 cells and facilitate their apoptosis through down-regulating the expression of NF-κB protein. HBX can accelerate the cell cycle of hepatic cancer HepG2 cells and suppress the apoptosis through up-regulating the expression of NF-κB protein.

国家自然科学基金资助项目（No.30672405）