中华神经医学杂志
中華神經醫學雜誌
중화신경의학잡지
CHINESE JOURNAL OF NEUROMEDICINE
2015年
6期
547-552
,共6页
伍健伟%梁建峰%何伟文%袁忠民
伍健偉%樑建峰%何偉文%袁忠民
오건위%량건봉%하위문%원충민
神经胶质瘤%曲古菌素A%JNK%细胞增殖
神經膠質瘤%麯古菌素A%JNK%細胞增殖
신경효질류%곡고균소A%JNK%세포증식
Glioma%Trichostatin A%JNK%Proliferation
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.
目的 探討組蛋白去乙酰化酶抑製劑抑製神經膠質瘤細胞株增殖的機製. 方法 分彆用0.1、0.2、0.5、1.0、2.0 μmol/L麯古菌素A(TSA)處理U251細胞48 h,0.5 μmol/L TSA處理細胞8、16、24、36、48 h,1μmol/L M344、0.5 μmol/L LBH589、6 mmol/L NaBu、6 mmol/L VPA處理細胞48 h,對照組加入等量溶劑,MTT法檢測細胞存活率;Western blotting檢測0.5 μmol/L TSA處理U251細胞8、16、24 h後c-Jun氨基末耑激酶(JNK)、燐痠化JNK (p-JNK)蛋白的錶達;Westernblotting、MTT法分彆檢測對照組、10 μmol/L SP600125組和1μmol/L CEP11004組細胞c-Jun、p-c-Jun蛋白的錶達和細胞存活率;Western blotting、MTT法分彆檢測轉染pcDNA3.1組、轉染pcDNA3.1 +TSA組、轉染pMKK7-JNK1組、轉染pMKK7-JNK1 +TSA組細胞p-c-Jun、Flag蛋白的錶達和細胞存活率. 結果 0.1、0.2、0.5、1.0、2.0 μmol/L TSA組細胞存活率低于對照組,且TSA濃度越高,細胞存活率越低,半數抑製濃度(IC50)為0.5μmol/L;0.5 μmol/L TSA處理16、24、36、48 h後細胞存活率低于對照組,且處理時間越長,細胞存活率越低;1 μmol/L M344、0.5 μmol/L LBH589、6 mmol/L NaBu、6 mmol/L VPA組細胞存活率低于對照組,差異均有統計學意義(P＜0.05).0.5 μmol/L TSA處理16h和24 h後細胞p-JNK蛋白水平顯著降低;10 μμmol/L SP600125、1μmol/L CEP11004組細胞p-c-Jun蛋白的錶達降低;10 μmol/L SP600125、1μmol/L CEP11004組細胞存活率低于對照組,差異有統計學意義(P＜0.05);與轉染pcDNA3.1組比較、轉染pMKK7-JNK1組細胞存活率增加,與轉染pcDNA3.1 +TSA組比較,轉染pMKK7-JNK1 +TSA組細胞存活率增加,差異有統計學意義(P＜0.05). 結論 組蛋白去乙酰化酶抑製劑抑製神經膠質瘤增殖的機製包含抑製JNK活性.
목적 탐토조단백거을선화매억제제억제신경효질류세포주증식적궤제. 방법 분별용0.1、0.2、0.5、1.0、2.0 μmol/L곡고균소A(TSA)처리U251세포48 h,0.5 μmol/L TSA처리세포8、16、24、36、48 h,1μmol/L M344、0.5 μmol/L LBH589、6 mmol/L NaBu、6 mmol/L VPA처리세포48 h,대조조가입등량용제,MTT법검측세포존활솔;Western blotting검측0.5 μmol/L TSA처리U251세포8、16、24 h후c-Jun안기말단격매(JNK)、린산화JNK (p-JNK)단백적표체;Westernblotting、MTT법분별검측대조조、10 μmol/L SP600125조화1μmol/L CEP11004조세포c-Jun、p-c-Jun단백적표체화세포존활솔;Western blotting、MTT법분별검측전염pcDNA3.1조、전염pcDNA3.1 +TSA조、전염pMKK7-JNK1조、전염pMKK7-JNK1 +TSA조세포p-c-Jun、Flag단백적표체화세포존활솔. 결과 0.1、0.2、0.5、1.0、2.0 μmol/L TSA조세포존활솔저우대조조,차TSA농도월고,세포존활솔월저,반수억제농도(IC50)위0.5μmol/L;0.5 μmol/L TSA처리16、24、36、48 h후세포존활솔저우대조조,차처리시간월장,세포존활솔월저;1 μmol/L M344、0.5 μmol/L LBH589、6 mmol/L NaBu、6 mmol/L VPA조세포존활솔저우대조조,차이균유통계학의의(P＜0.05).0.5 μmol/L TSA처리16h화24 h후세포p-JNK단백수평현저강저;10 μμmol/L SP600125、1μmol/L CEP11004조세포p-c-Jun단백적표체강저;10 μmol/L SP600125、1μmol/L CEP11004조세포존활솔저우대조조,차이유통계학의의(P＜0.05);여전염pcDNA3.1조비교、전염pMKK7-JNK1조세포존활솔증가,여전염pcDNA3.1 +TSA조비교,전염pMKK7-JNK1 +TSA조세포존활솔증가,차이유통계학의의(P＜0.05). 결론 조단백거을선화매억제제억제신경효질류증식적궤제포함억제JNK활성.
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.
