摘要
目的探讨组蛋白去乙酰化酶抑制剂Apicidin对胶质瘤细胞系U87Nanog和p53表达及细胞增殖、周期、凋亡的影响。方法体外常规培养U87细胞,MTT检测0.2、0.5、1.0、2.0ixmol/LApicidin处理24、48、72h后细胞抑制率的变化,同时设对照组,RT.PCR和Westernblotting法检测1.0μmol/LApicidin处理48h后细胞Nanog与p53mRNA和蛋白水平的改变。DAPI染色检测1.0μmol/LApicidin作用后细胞凋亡的改变;流式细胞仪检测0.2、0.5、1.0μmol/LApicidin作用48h后U87细胞凋亡率和细胞周期的变化。结果MTT显示不同浓度Apicidin处理后细胞生长出现抑制,并呈剂量、时间依赖性,48h细胞增殖的半数抑制浓度(IC茹为(1.74±0.13)μmol/L;RT.PCR检测发现1.0ixmoFLApicidin组细胞NanogmRNA表达较对照组降低,p53mRNA表达增高。Westernblotting检测显示与对照组比较,Nanog蛋白水平降低,而p53蛋白水平增高,差异有统计学意义(P〈0.05)。DAPI染色发现1.0μmol/LApicidin组细胞核出现凋亡表现;流式细胞仪检测发现,0.5、1.0μmol/LApicidin作用48h后U87细胞凋亡率为11.57%、19.67%,高于对照组(2.2%)。S期细胞比例分别为(42.92±0.56)%、(56.95±0.53)%,高于对照组的(32.68±0.49)%,差异有统计学意义(P〈0.05)。结论Apicidin可以显著抑制U87胶质瘤细胞系生长,诱导细胞周期阻滞和凋亡产生,其机制可能与抑制干细胞标志性基因Nanog的表达和抑癌基因p53的转录及蛋白表达增加有关。
Objective To investigate the effect of histone deacetylase inhibitor Apicidin on apoptosis, cycle, proliferation and Nanog and p53 expressions of U87 glioblastoma cell line. Methods U87 cells were cultured routinely in vitro; MTT assay was employed to detect the changes of cell inhibition after 0.2, 0.5, 1.0 and 2.0μmol/L Apicidin treatment; RT-PCR and Western blotting were employed to measure the mRNA and protein expressions of Nanog and p53 in cells of the Apicidin treatment group (treated with 1.0 μmol/L Apicidin for 48 h) and control group; DAPI staining was employed to observe the changes of cell apoptosis after being treated with 1.0 μmol/L Apicidin; cell cycle arrest and apoptosis rate were examined by ftow cytometry after being treated with 0.2, 0.5 and 1.0 μmol/L Apicidin for 48 h. Results MTT assay showed that the cell growth was inhibited after being treated with different doses of Apicidin, with dose- and time-dependent manners; the median inhibitory concentration (IC^o) of proliferation 48 h after treatment was approximately 1.74+0.13 ~mol/L. RT-PCR showed that 1.0 p^molfL Apicidin treatment group had significantly lower Nanog mRNA expression and higher p53 mRNA expression than control group (P〈0.05); Western blotting showed that 1.0 ixmol/L Apicidin treatment group had significantly lower Nanog protein expression and higher p53 protein expression than control group (P〈0.05). DAPI staining showed apoptotic nuclei in cells of 1.0 IJ mol/L Apicidin treatment group. Flow cytometry indicated that the apoptosis rate in the 0.5 and 1.0 ~zmol/L Apicidin treatment groups (11.57 % and 19.67 %) was significantly higher than that in the control group (2.2 %). The percentage of cells being arrested at S phase was 42.92~0.56% and 56.95~0.53% in the 0.5 and 1.0 p^mol/L Apicidin treatment groups, which was significantly higher than that in the control group (32.68+0.49%, P〈0.05). Condusion Histone deacetylase inhibitor Apicidin could sequentially repress the proliferation of glioma cell line U87 and induce cell cycle arrest and apoptosis in U87 cells, whose molecular mechanisms might relate to activate the tumor suppressor p53 expression and inhibit Nanog expression.
出处
《中华神经医学杂志》
CAS
CSCD
北大核心
2014年第3期246-251,共6页
Chinese Journal of Neuromedicine
基金
国家自然科学基金(81172407)
安徽省重点实验室绩效考核项目(1306c083028)