摘要
目的 探讨DNA依赖蛋白激酶（DNA-PK）抑制剂NU7026和渥曼青霉素（Wortmannin）对1,4-苯醌（1,4-BQ）诱导的人早幼粒白血病细胞（HL60）细胞凋亡的影响.方法 HL60细胞分为染毒组（0、5、10、25和50μmol/L1,4-BQ染毒24 h）和NU7026 、Wortmannin预处理组（10μmol/L NU7026、25μmol/L Wortmannin分别预处理1h后以0、5、10、25和50 μmol/L 1,4-BQ染毒24 h）,用流式细胞仪Annexin V/PI双染法和DNA Ladder法分析检测细胞凋亡水平.将HL60细胞分为空白对照组、NU7026处理组（10 μmol/L）、Wortmannin处理组（25 μmol/L）、1,4-BQ染毒组（10 μmol/L）、NU7026＋1,4-BQ组（10μmol/L NU7026预处理1h,以10 μmol/L 1,4-BQ染毒24 h）,25 μmol/L Wortmannin＋1,4-BQ组（25μmol/L Wortmannin预处理1h,以10 μmol/L 1,4-BQ染毒24 h）,用real-time PCR法检测Bax mRNA基因表达;蛋白免疫印迹法（Western blot）检测HL60细胞的p53蛋白表达.结果 流式细胞仪Annexin V/PI双染法结果显示,NU7026＋10 μmol/L 1,4-BQ处理组细胞凋亡率为17.6％±1.19％,Wortmannin＋ 10μmol/L 1,4-BQ处理组细胞凋亡率为15.2％±1.22％,两组细胞凋亡率均高于10 μmol/L 1,4-BQ染毒组（6.3％±1.04％）;NU7026＋25tμmol/L1,4-BQ处理组细胞凋亡率为46.2％±3.55％,Wortmannin＋25 μmol/L1,4-BQ处理组细胞凋亡率为26.9％±2.62％,两组细胞凋亡率均明显高于25 μmol/L 1,4-BQ染毒组（14.1％±1.54％）;NU7026＋50 μmol/L1,4-BQ处理组细胞凋亡率为61.8％±1.78％,明显高于50 μmol/L1,4-BQ染毒组（35.9％±4.51％）,以上各组的差异均有统计学意义（P＜0.05）.DNA Ladder法结果与流式细胞仪检测数据基本一致.与NU7026组和1,4-BQ染毒组比较,NU7026＋1,4-BQ组Bax mRNA表达水平升高;与Wortmannin组和1,4-BQ组比较,Wortmannin＋1,4-BQ组Bax mRNA表达水平升高,差异均有统计学意义（P＜0.05）.Western blot检测HL60细胞不表达p53蛋白.结论 DNA-PK抑制剂NU7026和Wortmannin促进1,4-BQ诱导的非p53依赖的HL60细胞�
Objective To investigate the impact of NU7026 and Wortmannin,inhibitors of DNA-dependent protein kinase （DNA-PK）,in HL60 cells apoptosis induced by 1,4-benzoquinone （1,4-BQ）.Methods HL60 cells were divided into three groups according to the exposures：the poisoned groups which were treated with 0,5,10,25 and 50 μmol/L 1,4-BQ for 24 h,respectively、the NU7026 groups which were preincubated with 10 μmol/L NU7026 for 1h prior to the 24h treatment of 0、5、10、25 and 50 μmol/L 1,4-BQand the Wortmannin groups which were preincubated with 25 μmol/L Wortmannin for 1h prior to the 24 h treatment of 0,5,10,25 and 50 μmol/L 1,4-BQ.Then we detected the apoptosis via flowcytometry Annexin V-FITC/PI and the DNA Ladder,respectively.We also tested the expressions of Bax mRNA with Real-Time PCR in HL60 cells which were exposed to 10 μmol/L NU7026 for 24 h,25 μmol/L Wortmannin 24 h,10 μmol/L 1,4-BQ 24 h,10 μmol/L NU7026 1h＋10 μmol/L 1,4-BQ 24 h and 25 μmol/L Wortmannin 1 h＋10 μ mol/L 1,4-BQ 24 h,as well as null （control）.We also examed the expressions of p53 in HL60 cells with Western blot.Results Annexin V-FITC/PI apoptosis tests suggested that apoptosis rates of NU7026＋10 μmol/L 1,4-BQgroup and Wortmannin ＋10 μmol/L 1,4-BQ were 17.6±1.19% and 15.2±1.22%,respectively.Both of results were higher than that of 10 μmol/L 1,4-BQ group （6.3±1.04%）; Apoptosis of NU7026＋25 μmol/L 1,4-BQ group was 46.2±3.55%,and Wortmannin ＋25 μmol/L 1,4-BQ group 26.9±2.62%.Both of results were also higher than that of 25μmol/L 1,4-BQ group （14.1±1.54%）; Apoptosis of NU7026＋50 μ mol/L 1,4-BQ group （61.8±1.78%） was higher than that of 50 μmol/L 1,4-BQ group （35.9±4.51%）.The above results were all statistically significant （P〈0.05）.Results of DNA-Ladder were basically consistent with those of Annexin V-FITC/PI apoptosis tests.In addition,both NU7026 and Wortmannin pretreatment elicited the higher expression of Bax mRNA in HL60 treated by 1,4-benzoquinone with statistically sig
出处
《中华劳动卫生职业病杂志》
CAS
CSCD
2015年第1期20-23,共4页
Chinese Journal of Industrial Hygiene and Occupational Diseases
基金
国家自然科学基金项目（81072276）
南昌市科技计划项目（2011-ZCSF-YLWSYRKJK-038）
关键词
蛋白激酶类
酶抑制剂
苯醌类
Protein kinases
Enzyme inhibitors
Ben zoquinones
