三氮唑席夫碱衍生物对人肝癌细胞分化和凋亡的影响

Effects of triazole schiff base derivative on the differentiation and apoptosis of human hepatocarcinoma cells

目的探讨三氮唑席夫碱衍生物对人肝癌细胞BEL-7402的诱导分化和凋亡作用。方法用不同剂量的3-吡啶-3-基-4[（4-羟基-3-甲氧基-苯亚甲基）氨基]-5-甲硫基-1，2，4-三唑（LH-37）与BEL-7402细胞共同培养，以MTT法检测细胞增殖和LH-37对细胞增殖的抑制作用；RT-PCR方法检测细胞甲胎蛋白（α-FP）和白蛋白（Alb）mRNA表达；ELISA法测定细胞仅．FP含量；免疫组织化学检测细胞内激活型Caspase-3表达；Westernblot检测Caspase-3和Caspase-9蛋白表达；可见光法测定超氧化物歧化酶（SOD）和过氧化氢酶（CAT）活力；荧光显微镜观察凋亡细胞形态；Annexin—V和PI双染流式细胞术测定凋亡细胞数。结果LH-37在10μmol·L^-1mmol·L^-1的浓度范围能抑制细胞增殖，且抑制率随浓度的增加而增高（P〈0．05或P〈0．01），10μmol·L^-1和1．0μmol·L^-1的LH-37使细胞AlbmRNA表达量增高，α-FPmRNA和蛋白质表达量明显下降，Caspase-3和Caspase-9表达量明显增加，Caspase-3阳性细胞明显增加；药物浓度达100μmol·L^-1作用48h后，BEL-7402细胞皱缩，呈现凋亡各期改变，细胞凋亡率高于对照组（P〈0．05）。结论三氮唑席夫碱衍生物对人肝癌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。

Aim To study the effect of 3-pyridin-3-yl- 4- [ （ 4-hydroxy-3-methoxy-benzylidene ） amino ] -5-methylsulfanyl4H-[ 1,2,4 ] triazole （ LH-37 ） on induction of differentiation and apoptosis of hepatocarcinoma BEL-7402 cells. Methods BEL-7402 cells were cultured in RPMI 1640 and treated by LH-37 at different doses. The proliferation of the cells and the inhibition effect of LH-37 on the cell proliferation were examined by MTr assay. The reverse transcriptase-polymerase chain reaction （RT-PCR） was used to detect the changes of alpha fetoprotein （α-FP）mRNA and albumin （Alb）. The concentration of α-FP in the cells was detected by ELISA assay. Cell morphology was observed by fluorescence microscope techniques. The protein expressions of active caspase-3 in BEL-7402 cells were observed by immunohistochemical staining. Western blot was used to assay caspase-3 and caspase-9. Colorimetric method was used to assay activity of superoxide dismutase （SOD） and catalase （CAT）. The apoptotic cells were assayed by flow cytometry （FCM） with annexin V-FITC conjugated and propidium iodide （PI）staining. Results The cell proliferation was inhibited by LH-37 at 10 μmol·L^-1 mmol·L^-1 in dose dependent manners. After treated with 10 μmol·L^-1 or 1.0 μmol·L^-1 LH-37, the mRNA and protein expression of α-FP were significantly reduced but mRNA expression of Alb was significantly increased. Treatment of BEL-7402 cells with different LH-37 concentrations for 48 hours increased the percentage of active caspase- 3 positive cells and protein expression of caspase-3 and caspase-9. More apoptosis features of cells were observed in LH-37 （ 100 μmol·L^-1 ） treatment groups than in control group. LH-37 markedly promoted the viability of SOD and decreased CAT in BEL-7402 cells. Counelusion LH-37 might inhibit proliferation and induce differentiation and apoptosis of human hepatocarcinoma BEL-7402 cells, which might be related to the cytotoxicity of the intracellular hydrogen peroxide （H2O2）.