论文部分内容阅读
目的探讨曲古抑菌素A(Trichostatin A,TSA)联合肿瘤坏死因子(tumor necrosis factor,TNF)相关凋亡诱导配体(TNF related apoptosis inducing ligand,TRAIL)对肝癌细胞Bel7402增殖凋亡的影响及其机制。方法采用四甲基偶氮唑盐(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的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。结果不同浓度TSA作用6、12和24 h对人肝癌Bel7402细胞的增殖没有明显抑制作用,而作用48 h后的细胞增殖抑制率明显升高,和对照组比较差异有统计学意义(P<0.05);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治疗的敏感度,TSA预处理联合TRAIL(100ng/ml)作用细胞24 h后,细胞生存率为(57.1±5.4)%,和单独药物处理组及对照组比较差异有统计学意义(P<0.05);DAPI染色显示TSA和TRAIL联合作用后Bel7402细胞有核凋亡出现。荧光显微镜观察证明单独应用TSA(200 ng/ml)或TRAIL(100 ng/ml)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的敏感度;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。
Objective To investigate the effect of trichostatin A (TSA) combined with tumor necrosis factor (TNF) -related apoptosis inducing ligand (TRAIL) on proliferation and apoptosis of hepatocellular carcinoma cell line Bel7402, Its mechanism. Methods MTT method was used to detect the growth inhibition rate of Bel7402 cells treated with TSA, TRAIL and low concentrations of TSA combined with TRAIL. The inhibitory rates of 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining was used to observe the apoptotic morphology of the cells treated with the drugs. Immunocytochemistry and Western blotting were used to observe the expression and localization of p65 protein in the cells. Results After treated with different concentrations of TSA for 6, 12 and 24 h, the proliferation of Bel7402 cells was not significantly inhibited, while the inhibition rate of cells proliferation after 48 h was significantly higher than that of the control group (P <0.05) ). The survival rate of Bel7402 cells treated with different concentrations of TRAIL did not change significantly. Pretreatment with low concentration of TSA (20 ng / ml) increased the sensitivity of Bel7402 cells to TRAIL treatment. TSA pretreatment combined with TRAIL (100ng / ml) (57.1 ± 5.4)%. Compared with the drug-treated group and the control group, the difference was statistically significant (P <0.05). The DAPI staining showed that the nuclear apoptosis of Bel7402 cells occurred after the combination of TSA and TRAIL. Fluorescence microscopy showed that the p65 protein in cells treated with TSA alone (200 ng / ml) or TRAIL (100 ng / ml) had some nuclear translocation, and the combination of the two drugs resulted in a decrease of p65 protein expression and significant Nuclear transfer and aggregation. Conclusion TSA at low concentration can increase the sensitivity of Bel7402 cells to TRAIL. The mechanism may be that the combination of two drugs can decrease the expression and activity of p65 and induce the apoptosis of Bel7402 cells.