摘要：
Objective To investigate the mechanism of histone deacetylase inhibitors in proliferation ofglioma cells.Methods (1) Glioma cell line U251 was cultured in vitro and treated with Trichostatin A (TSA) at 0.1,0.2,0.5,1.0 or 2.0 μmol/L for 48 h to determine the IC50 for TSA,and then,cells were treated with TSA at the IC50 concentration (0.5 μ mol/L) for 8,16,24,48 h;1 μmol/L M344,0.5 μmol/L LBH589,6 mmol/L NaBu and 6 mmol/L VPA were used to treat the cells for 48 h and same volume of solvent was given to cells for 48 h as control group;MTT assay was performed to determine cell viability.(2) Western blotting was performed to test the Jun N-terminal kinase (JNK)expression and phosphorylated JNK (p-JNK) level in the U251 cells after being treated with 0.5 μmol/L TSA for 8,16 and 24 h.(3) Western blotting and MTT assay were employed to detect the c-Jun expression and phosphorylated c-Jun (p-c-Jun) level,and cell viability in the U251 cells of control group,10 μmol/L SP600125 treatment group (JNK inhibitor) and 1 μmol/L CEP11004 treatment group (MLK3,a direct upstream kinase of JNK).(4) Western blotting and MTT assay were employed to detect the c-Jun and Flag expressions,and cell viability in the U251 cells of pcDNA3.1 transfected group,pcDNA3.1+TSA transfected group,pMKK7-JNK1 transfected group and pMKK7-JNK1+TSA transfected group.Results (1) The viability of cells of 0.1,0.2,0.5,1.0 or 2.0 μmol/L TSA treated group was significantly lower than that in the control group,and the higher the TSA concentration,the lower the viability of cells;the viability of cells of 0.5 μmol/L TSA treated for 16,24,36 and 48 h groups was significantly lower than that in the control group,and the longer the TSA treatment,the lower the viability of cells.(2) The viability of 1 μmol/L M344,0.5 μmol/L LBH589,6 mmol/L NaBu and 6 mmol/L VPA treatment groups was significantly lower than that in the control group (P＜0.05).(3) As compared with those in the control group,the p-JNK expression was significantly decreased in the cells after 0.5 μmol/L TSA treatment for 16 and 24 h,and the p-c-Jun protein expression and the cell viability were significantly decreased in the cells of 10 μmol/L SP600125 and 1 μmol/L CEP11004 treatment groups (P＜0.05).(4) As compared with that in the pcDNA3.1 transfected group,cell viability in the U251 cells ofpcDNA3.1+TSA transfected group,pMKK7-JNK1 transfected group and pMKK7-JNK1+TSA transfected group was significantly increased(P＜0.05).Conclusion Histone deacetylase inhibitors reduce the proliferation of glioma cell U251 through inhibiting JNK activity.%目的 探讨组蛋白去乙酰化酶抑制剂抑制神经胶质瘤细胞株增殖的机制. 方法 分别用0.1、0.2、0.5、1.0、2.0 μmol/L曲古菌素A(TSA)处理U251细胞48 h,0.5 μmol/L TSA处理细胞8、16、24、36、48 h,1μmol/L M344、0.5 μmol/L LBH589、6 mmol/L NaBu、6 mmol/L VPA处理细胞48 h,对照组加入等量溶剂,MTT法检测细胞存活率;Western blotting检测0.5 μmol/L TSA处理U251细胞8、16、24 h后c-Jun氨基末端激酶(JNK)、磷酸化JNK (p-JNK)蛋白的表达;Westernblotting、MTT法分别检测对照组、10 μmol/L SP600125组和1μmol/L CEP11004组细胞c-Jun、p-c-Jun蛋白的表达和细胞存活率;Western blotting、MTT法分别检测转染pcDNA3.1组、转染pcDNA3.1 +TSA组、转染pMKK7-JNK1组、转染pMKK7-JNK1 +TSA组细胞p-c-Jun、Flag蛋白的表达和细胞存活率. 结果 0.1、0.2、0.5、1.0、2.0 μmol/L TSA组细胞存活率低于对照组,且TSA浓度越高,细胞存活率越低,半数抑制浓度(IC50)为0.5μmol/L;0.5 μmol/L TSA处理16、24、36、48 h后细胞存活率低于对照组,且处理时间越长,细胞存活率越低;1 μmol/L M344、0.5 μmol/L LBH589、6 mmol/L NaBu、6 mmol/L VPA组细胞存活率低于对照组,差异均有统计学意义(P＜0.05).0.5 μmol/L TSA处理16h和24 h后细胞p-JNK蛋白水平显著降低;10 μμmol/L SP600125、1μmol/L CEP11004组细胞p-c-Jun蛋白的表达降低;10 μmol/L SP600125、1μmol/L CEP11004组细胞存活率低于对照组,差异有统计学意义(P＜0.05);与转染pcDNA3.1组比较、转染pMKK7-JNK1组细胞存活率增加,与转染pcDNA3.1 +TSA组比较,转染pMKK7-JNK1 +TSA组细胞存活率增加,差异有统计学意义(P＜0.05). 结论 组蛋白去乙酰化酶抑制剂抑制神经胶质瘤增殖的机制包含抑制JNK活性.
