【作者】 李伟； 朱明月； 董栩； 夏华； 陈栘； 鲁琰； 谢协驹； 符史干； 李孟森；

【Author】 LI Wei;ZHU Mingyue;DONG Xu;XIA Hua;CHEN Yi;LU Yan;GUO Junli;XIE Xieju;FU Shigan;LI MengSeng;Hainan Provincial Key Laboratory of Carcinogenesis and Intervention,Hainan Medical College;Hainan Key Laboratory of Molecular Biology,Hainan Medical College;Department of Pathophysiology,Hainan Medical College;Department of Physiology,Hainan Medical College;Institution of Tumor,Hainan Medical College;

【机构】 海南医学院肿瘤发生和干预重点实验室； 海南医学院分子生物学重点实验室； 海南医学院病理生理学教研室； 海南医学院； 海南医学院生理学教研室； 海南医学院肿瘤研究所；

【摘要】 目的探讨曲古抑菌素A(Trichostatin A,TSA)联合肿瘤坏死因子相关凋亡诱导配体(TNF related apoptosis inducing ligand,TRAIL)对肝癌细胞Bel7402增殖凋亡的影响,并初步探讨其可能机制。方法采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)染色法分别检测TSA、TRAIL及低浓度TSA联合TRAIL处理Bel7402细胞的生长抑制率;4,6-二脒基-2-苯基吲哚二盐酸盐(DAPI)染色法对药物联合处理后细胞进行凋亡形态学观察;免疫细胞化学和Western blot技术观察药物联合作用后p65蛋白在细胞中表达和定位的变化。结果MTT结果显示,不同浓度TSA作用6h、12h、24h对人肝癌细胞的增殖没有明显抑制作用,而作用48h后细胞增殖抑制率明显升高,呈一定的时间依赖性;不同浓度TRAIL处理Bel7402细胞存活率没有明显改变;低浓度TSA(20 ng/ml)预处理能够增加Bel7402细胞对TRAIL治疗的敏感性,与对照组相比,TSA预处理联合TRAIL(100ng/ml)作用细胞24h后,细胞的增殖抑制率和单独药物处理组比较有统计学差异(p<0.05);单独应用TSA或TRAIL处理的细胞p65蛋白有部分核内移位,而两种药物联合应用导致p65蛋白表达量降低,并且发生明显的核内转移和集聚。结论低浓度TSA能够增加肝癌Bel7402细胞对TRAIL的敏感性;其机制可能是两种药物联合应用降低p65的表达和活性,从而诱导Bel7402细胞凋亡。更多还原

【Abstract】 Objective To investigate the effect of Trichostatin A(TSA) synergy tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) on proliferation and apoptosis in human hepatocellular carcinoma cell line Bel-7402 in vitro,preliminary explore the possible regulation mechanism of this effect.Methods The effect of TSA or TRAIL,and TSA synergy TRAIL on proliferation of Bel 7402 cells was tested by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) stained method,4,6-diamidino-2-phenylindole dihydrochlorid(DAPI) staining method was applied to display the apotosome morphology of hepatocellular carcinoma cells;the expression and subcellular location of p65 was investigated by immuno-cytochemistry and Western blot.Results MTT analysis indicated that there were no influence on the growth of Bel 7402 cells when treated with different concentrations of TSA for 6,12 or 24 h,but proliferation of Bel7402 cells were significant inhibitory while treated with 50 or100 ng/ml TSA for 48 h;The proliferation of Bel7402 cells also has little changed while treated with TRAIL for 6 h,12 h or 24 h;Low-concentration of TSA(20 ng/ml) was pretreated followed treated with TRAIL been able to restrain the growth of Bel7402 cells significantly,the growth inhibiting ratio of Bel7402 cells has statistical different,P<0.05 vs control group.DAPI staining method indicated that Bel7402 cells trended to characteristic morphological changes of apoptosis when treated by TSA combined with TRAIL.Fluorescence microscope and Western blot confirmed that the expression and location of p65 presented dominantly in cytoplasm;However,the translocation of p65 from the cytoplasm into the nucleus while treated with TSA(200 ng/ml)or TRAIL(100 ng/ml) for 24 h,and the translocation and accumulation of p65 from cytoplasm to nucleus and the expression of p65 were repressed when treated with low concentration of TSA combined with TRAIL.Conclusion TSA has a capacity to enhance TRAIL-induced apoptosis of hepatocellular carcinoma Bel7402 cells;this effect probably involve in decreasing the expression and activity of p65 of Bel7402 cells.更多还原