肝癌组织NF-κB表达及活化干预对肝癌细胞增殖的影响

Intervention of NF-κB activation inhibits the growth of human hepatocellular carcinoma HepG2 cells

目的:分析核转录因子-κB(NF-κB)表达及活化干预对肝癌细胞HepG2增殖的影响.方法:培养HepG2肝癌细胞并给予抗人肿瘤坏死因子(TNF)-α单克隆抗体,以流式细胞术和Annexin V-FITC/PI双色标记法分别检测细胞周期时相和细胞凋亡的变化;以酶联免疫吸附法(ELISA)定量分析肝癌组织及用药前后细胞培养液中TNF-α和核蛋白NF-κB的变化.结果:人肝癌的癌灶组织中NF-κB表达,较癌旁组织明显增强(P<0.01);HepG2细胞培养给予抗TNF-α单克隆抗体(5mg/L)后,肝癌细胞凋亡率明显高于对照(21.45%±4.07%vs5.63%±0.93%,q=10.07,P<0.01);G0/G1明显高于对照(66.23%±1.29%vs59.00%±1.02%,q=10.98,P<0.01),而S期细胞比例无明显改变;用药后细胞株NF-κB水平明显低于对照(59.00ng/mg±1.02ng/mg核蛋白vs73.88ng/mg±7.41ng/mg核蛋白,q=18.92,P<0.01),与培养液中明显降低的TNF-α水平呈显著正相关(r=0.89,P<0.01),抑制效应呈浓度依赖性,高浓度时作用最明显(P<0.01).结论:以TNF-α抗体干预NF-κB信号通路活化,可使肝癌细胞凋亡增加,细胞周期阻滞在G0/G1期,肝癌细胞增殖明显抑制.

AIM：To investigate the impact of intervention of nuclear factor-κB（NF-κB） activation with tumor necrosis factor-α（TNF-α） monoclonal antibody（TNF-α mab） on the proliferation of human hepatocellular carcinoma（HCC） HepG2 cells.METHODS：HepG2 cells were cultured in vitro and incubated with TNF-α mab.The changes in cell cycle and apoptosis were detected by flow cytometry（FCM） and annexin-V/PI double staining assay,respectively.The expression of NF-κB and TNF-α in human liver cancer,tumor-adjacent liver tissue,and HepG2 cells were quantitatively analyzed by enzyme-linked immunosorbent assay（ELISA）.RESULTS：The expression level of NF-κB in human HCC was higher than that in tumoradjacent liver tissue（P 0.01）.The percentage of apoptotic cells in HepG2 cells treated with TNF-α mab（5 mg/L） was higher than that in untreated HepG2 cells（21.45% ± 4.07% vs 5.63% ± 0.93%,q = 10.07,P 0.01）.The percentage of cells in G 0 /G 1 phase was significantly higher in HepG2 cells treated with TNF-α mab than in untreated HepG2 cells（q = 10.98,P 0.01） though no significant difference was noted in the percentage of cells in S phase between the two groups of cells.The level of NF-κB in HepG2 cells treated with TNF-α mab was lower than that in untreated HepG2 cells [59.00 ng/mg ± 1.02 ng/mg nuclear protein（NP） vs 73.88 ng/mg ± 7.41 ng/mg NP,q=18.92,P0.01].Increased NF-κB level is correlated with decreased TNF-α level in HepG2 cells treated with TNF-α mab（r = 0.89,P 0.01）.The inhibitory effect of TNF-α mab on TNF-α level is dose-dependent（P 0.01）.CONCLUSION：Intervention of NF-κB activation by TNF-α mab inhibits the proliferation of HepG2 cells by inducing apoptosis and blocking the cells in G 0 /G 1 phase.