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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G0/G1期细胞比例显著增加,S期和G2/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G0/G1期细胞比例显著减少,S期和G2/M期细胞比例增加。PDTC作用后,L02/HBX/PDTC组凋亡率[(40.33±0.07)%]及G0/G1期细胞比例较L02/HBX组显著增加,G2/M期细胞比例明显减少,而HepG2/HBX/PDTC组凋亡率[(5.45±0.07)%]及G0/G1期细胞比例较HepG2/HBX组显著增加,S期和G2/M期细胞比例明显减少,但其凋亡率仍低于对照HepG2细胞。West-ern 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 induction of hepatitis B virus X protein (HBX) on the apoptosis of different hepatocytes by NF-κB signaling pathway. Methods: Human normal liver cell line L02 (L02 / HBX) and human hepatoma cell line HepG2 (HepG2 / HBX) stably transfected with PEGFP-N1-HBX plasmid were established and treated with a specific NF- NF-κB signal pathway was blocked by pyrrolidine dithiocarbamate (PDTC) and the cell cycle and apoptosis of L02 and HepG2 cells before and after PEGFP-N1-HBX transfection were detected by flow cytometry. The expressions of NF- κB expression. Results: L02 / HBX and HepG2 / HBX cells stably transfected with PEGFP-N1-HBX were successfully constructed. Compared with L02 cells in control group, the apoptosis rate of L02 / HBX cells was significantly increased [(31.31 ± 0.51)% vs (14.05 ± 0.09)%, P <0.05]; the proportion of cells in G0 / G2 / M phase cells decreased. The apoptosis rate of HepG2 / HBX cells was significantly lower than that of HepG2 cells in control group [(1.21 ± 0.04)% vs (10.26 ± 0.10)%, P <0.05]. The percentage of cells in G0 / G2 / M phase cells increased. After PDTC treatment, the percentage of cells in L02 / HBX / PDTC group [(40.33 ± 0.07)%] and G0 / G1 phase was significantly increased compared with L02 / HBX group, while the proportion of cells in G2 / The percentage of apoptosis in PDTC group [(5.45 ± 0.07)%] and G0 / G1 phase was significantly higher than that in HepG2 / HBX group, and the percentage of cells in S phase and G2 / M phase was significantly decreased, but the apoptosis rate was still lower than that of control HepG2 cell. West-ern blotting results showed that the expression of NF-κB in L02 / HBX cells was significantly down-regulated, whereas the expression of NF-κB in HepG2 / HBX cells was significantly increased. The expression of NF-κB in L02 / HBX / PDTC and HepG2 / HBX / Conclusion: HBX can down-regulate the expression of L02 NF-κB in normal liver cells, arrest the cell cycle and promote the apoptosis of cells. HBX can increase the expression of NF-κB in HepG2 and accelerate the cell cycle Death.