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目的观察大茴香醛缩氨基均三唑席夫碱对人肝癌细胞BEL-7402的诱导分化和凋亡作用。方法用不同浓度的3(3-吡啶)-4-[(4-甲氧基-苯亚甲基)氨基]-5-甲硫基-1,2,4-三唑(LH-38)与BEL-7402细胞体外培养。四甲基偶氮唑蓝(MTT)法检测细胞增殖和LH-38对细胞增殖的抑制作用。用Hoechst 33258荧光染色细胞形态学和Annexin V/PI双标记技术观察细胞凋亡变化。放射免疫法检测BEL-7402细胞甲胎蛋白(α-FP)和白蛋白(Alb)的分泌量,以了解细胞分化状态。蛋白质免疫印迹(Western blotting)半定量分析Caspase-3和p53的表达。可见光法测定超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活力。结果LH-38在0.1~10μmol·L-1的浓度范围能抑制细胞增殖,且抑制率随浓度和时间的增加而增高。10和1.0μmol·L-1的LH-38使细胞Alb、Caspase-3和p53的蛋白表达明显增加,α-FP含量下降。SOD活性升高(P<0.01),而CAT活性降低,差异有显著性(P<0.01,P<0.05)。药物浓度在10μmol·L-1作用48h,BEL-7402细胞皱缩,呈现凋亡各期改变,细胞凋亡率显著高于对照组(P<0.05)。结论大茴香醛缩氨基均三唑席夫碱对人肝癌BEL-7402细胞具有抗增殖、促分化和凋亡作用,作用可能与过氧化氢的氧化损伤作用有关。
Objective To observe the induction and differentiation of human hepatocellular carcinoma cell line BEL-7402 induced by anisaldehyde-aminomethylated Schiff base. Methods Different concentrations of 3-pyridyl-4 - [(4-methoxy-benzylidene) amino] -5-methylthio-1,2,4-triazole (LH- BEL-7402 cells were cultured in vitro. Tetramethylbenzothiazolyl tetrazolium (MTT) method was used to detect the cell proliferation and the inhibitory effect of LH-38 on cell proliferation. Cell apoptosis was observed by Hoechst 33258 fluorescent staining and Annexin V / PI double labeling. The levels of α-FP and Alb in BEL-7402 cells were detected by radioimmunoassay to understand the cell differentiation status. Semi-quantitative analysis of Caspase-3 and p53 expression by Western blotting. The activities of superoxide dismutase (SOD) and catalase (CAT) were measured by visible light method. Results LH-38 inhibited cell proliferation in the concentration range of 0.1 ~ 10μmol·L-1, and the inhibition rate increased with the increase of concentration and time. LH-38 at 10 and 1.0 μmol·L-1 increased the protein expression of Alb, Caspase-3 and p53, and decreased the content of α-FP. SOD activity increased (P <0.01), while CAT activity decreased, the difference was significant (P <0.01, P <0.05). After treated with 10μmol·L-1 for 48h, the apoptosis of BEL-7402 cells was observed and the apoptotic rate was significantly higher than that of the control group (P <0.05). Conclusion Anisodamine trimethylazole Schiff base can inhibit the proliferation, differentiation and apoptosis of human hepatocellular carcinoma BEL-7402 cells, which may be related to the oxidative damage of hydrogen peroxide.