摘要目的 探讨干预肿瘤坏死因子(TNF)α/核因子-κ B(NF κ B)信号通路活化对肝癌细胞增殖的影响与分子机制.方法 给予雄性Sprague-Dawly大鼠0.05％的2乙酰氨基芴以制备肝癌模型;收集肝病患者外周血,分离血清和有核细胞;培养肝癌细胞HepG2,以TNFα抗体下调TNFα或以NF-κ B/p65siRNA干扰NF-κB基因转录.定量PCR检测NF κBmRNA,流式细胞术检测细胞周期,Annexin V-异硫氰酸/碘化丙啶双色标记法分析细胞凋亡,以Western blot或酶联免疫吸附法分析TNFα和NF-κB浓度.计量资料采用t检验、方差分析;计数资料以x2检验或Fisher确切概率法分析.结果 肝细胞恶性转化时TNF α/NF-κB信号通路中关键分子TNFα与NF-κB呈进行性升高,其水平与肝脏病理学改变呈正相关;从良性肝病进展到肝癌过程中,两者呈进行性升高趋势.HepG2细胞中加入TNF α抗体(5mg/L)后,细胞凋亡率(21.45％±4.07％)明显上升,显著高于对照组(5.63％±0.93％,q=10.07,P＜0.01);细胞增殖阻滞在G0／G1期,其比例(66.23％±1.29％)明显高于对照组(59.00％±1.02％,q=10.98,P＜0.01);且培养液中TNF α和NF-κB表达水平均显著下调(r=0.89,P＜0.01);抑制肝癌细胞效应与抗TNFα浓度呈剂量依赖性.肝癌细胞NF-κB高表达,显著高于L02细胞,以p65 siRNA(100 nmol/L)干预后,NF-κB在mRNA或蛋白水平分别下降93％或62％,且85％癌细胞发生凋亡.结论 干预TNFα/NF-κB信号通路活化,可经促凋亡和细胞周期阻滞机制抑制肝癌细胞增殖.

abstractsObjective To investigate the inhibitory effects of intervention of the tumor necrosis factor-alpha (TNFα)/nuclear factor-kappa B (NF-κB) signaling pathway activation on hepatoma cell proliferation and to explore its mechanism.Methods A rodent hepatoma model was established by feeding N-2-fluorenylacetamide (2-N-FAA) to male Sprague-Dawley rats.Human subjects with various liver diseases were enrolled in the study,and serum and peripheral blood nuclear cells were collected for analysis.HepG2 cells were cultured in vitro and treated with anti-TNFα (monoclonal antibody,mAb) to down-regulate its expression or transfected with siRNA targeting the p65 subunit of NF-κB to inhibit its activation.The liver cell line L02 was used as a control.Changes in protein and gene expression levels of NF-κB and TNFα were analyzed by Western blotting or enzyme-linked immunosorbent assay and real-time PCR,respectively.Changes in the cell cycle or apoptosis were evaluated by flow cytometry or Annexin-V/PI double-labeling assay,respectively.Results TNFα and NF-κB expression showed increasing trends during the malignant transformation of rat hepatocytes,and the differential expression patterns showed association with histopathological alterations in the hepatocytes.Following treatment with the TNFα mAb,the HepG2 cells showed a higher percentage of apoptotic cells than the untreated control cells (21.45％ ± 4.07％ vs.5.63％ ± 0.93％,q =10.07,P ＜ 0.01).There was a significant difference in the rate of cells in the G0/G1 phase in the p65-siRNA transfected cells (66.23％ ± 1.29％ vs.untreated control cells:59.00％ ± 1.02％,q =10.98,P ＜ 0.01).The decreased expression of TNFα and NF-κB in cell culture supematants was positively correlated with the dose of treatment (r =0.89,P ＜ 0.01),with the most robust decreases being achieved with the highest concentrations (P ＜ 0.01).NF-κB expression was significantly higher in the HepG2 cells than in the L02 cells,and transfection of p65-siRNA reduced the mRNA (93％) and protein (62％) levels and increased the cell apoptosis index (to 85％).Conclusion Proliferation of hepatoma cells may be significantly inhibited by intervening in the activation of the TNFα/NF-κB signaling pathway,which promotes cell apoptosis and blocks cell cycling.