摘要
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 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 （LO2/HBX or HepG2/HBX cells）. NF-κB signaling pathway inhibitor pyr rolidine 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 ceils and HepG2/HBX ceils 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 G0/G1 stage increased with cells in S and GJM stage decreased. The apoptosis of HepG2/HBX cells significantly decreased compared with the control HepG2 cells （[ 1.21 ±0.04]% vs [10. 26 ±0. 101%, P 〈 0.05 ）, and the proportion of cells in G0/G1 stage decreased with cells in S and GJM stage increased. After PDTC treatment, the proportion of L02/HBX/PDTC cells in G0/G~ phase increased significantly （[40.33 ±0.07 % ）, while that in S and GJM 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 Gn/G, phase of HeDG2/HBX/PDTC were in-creased significantly, and decreased remarkably in S and G2/M phase, while the apoptosis rate was still lower than the HepG2 cells. The expression of NF-κB protein was significantly decreased in EO2/HBX cells but increased in HepG2/HBX ceils 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.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2012年第4期402-407,共6页
Chinese Journal of Cancer Biotherapy
基金
国家自然科学基金资助项目(No.30672405)~~
