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细胞凋亡.
目的 探討DNA依賴蛋白激酶(DNA-PK)抑製劑NU7026和渥曼青黴素(Wortmannin)對1,4-苯醌(1,4-BQ)誘導的人早幼粒白血病細胞(HL60)細胞凋亡的影響.方法 HL60細胞分為染毒組(0、5、10、25和50μmol/L1,4-BQ染毒24 h)和NU7026 、Wortmannin預處理組(10μmol/L NU7026、25μmol/L Wortmannin分彆預處理1h後以0、5、10、25和50 μmol/L 1,4-BQ染毒24 h),用流式細胞儀Annexin V/PI雙染法和DNA Ladder法分析檢測細胞凋亡水平.將HL60細胞分為空白對照組、NU7026處理組(10 μmol/L)、Wortmannin處理組(25 μmol/L)、1,4-BQ染毒組(10 μmol/L)、NU7026+1,4-BQ組(10μmol/L NU7026預處理1h,以10 μmol/L 1,4-BQ染毒24 h),25 μmol/L Wortmannin+1,4-BQ組(25μmol/L Wortmannin預處理1h,以10 μmol/L 1,4-BQ染毒24 h),用real-time PCR法檢測Bax mRNA基因錶達;蛋白免疫印跡法(Western blot)檢測HL60細胞的p53蛋白錶達.結果 流式細胞儀Annexin V/PI雙染法結果顯示,NU7026+10 μmol/L 1,4-BQ處理組細胞凋亡率為17.6％±1.19％,Wortmannin+ 10μmol/L 1,4-BQ處理組細胞凋亡率為15.2％±1.22％,兩組細胞凋亡率均高于10 μmol/L 1,4-BQ染毒組(6.3％±1.04％);NU7026+25tμmol/L1,4-BQ處理組細胞凋亡率為46.2％±3.55％,Wortmannin+25 μmol/L1,4-BQ處理組細胞凋亡率為26.9％±2.62％,兩組細胞凋亡率均明顯高于25 μmol/L 1,4-BQ染毒組(14.1％±1.54％);NU7026+50 μmol/L1,4-BQ處理組細胞凋亡率為61.8％±1.78％,明顯高于50 μmol/L1,4-BQ染毒組(35.9％±4.51％),以上各組的差異均有統計學意義(P＜0.05).DNA Ladder法結果與流式細胞儀檢測數據基本一緻.與NU7026組和1,4-BQ染毒組比較,NU7026+1,4-BQ組Bax mRNA錶達水平升高;與Wortmannin組和1,4-BQ組比較,Wortmannin+1,4-BQ組Bax mRNA錶達水平升高,差異均有統計學意義(P＜0.05).Western blot檢測HL60細胞不錶達p53蛋白.結論 DNA-PK抑製劑NU7026和Wortmannin促進1,4-BQ誘導的非p53依賴的HL60細胞凋亡.
목적 탐토DNA의뢰단백격매(DNA-PK)억제제NU7026화악만청매소(Wortmannin)대1,4-분곤(1,4-BQ)유도적인조유립백혈병세포(HL60)세포조망적영향.방법 HL60세포분위염독조(0、5、10、25화50μmol/L1,4-BQ염독24 h)화NU7026 、Wortmannin예처리조(10μmol/L NU7026、25μmol/L Wortmannin분별예처리1h후이0、5、10、25화50 μmol/L 1,4-BQ염독24 h),용류식세포의Annexin V/PI쌍염법화DNA Ladder법분석검측세포조망수평.장HL60세포분위공백대조조、NU7026처리조(10 μmol/L)、Wortmannin처리조(25 μmol/L)、1,4-BQ염독조(10 μmol/L)、NU7026+1,4-BQ조(10μmol/L NU7026예처리1h,이10 μmol/L 1,4-BQ염독24 h),25 μmol/L Wortmannin+1,4-BQ조(25μmol/L Wortmannin예처리1h,이10 μmol/L 1,4-BQ염독24 h),용real-time PCR법검측Bax mRNA기인표체;단백면역인적법(Western blot)검측HL60세포적p53단백표체.결과 류식세포의Annexin V/PI쌍염법결과현시,NU7026+10 μmol/L 1,4-BQ처리조세포조망솔위17.6％±1.19％,Wortmannin+ 10μmol/L 1,4-BQ처리조세포조망솔위15.2％±1.22％,량조세포조망솔균고우10 μmol/L 1,4-BQ염독조(6.3％±1.04％);NU7026+25tμmol/L1,4-BQ처리조세포조망솔위46.2％±3.55％,Wortmannin+25 μmol/L1,4-BQ처리조세포조망솔위26.9％±2.62％,량조세포조망솔균명현고우25 μmol/L 1,4-BQ염독조(14.1％±1.54％);NU7026+50 μmol/L1,4-BQ처리조세포조망솔위61.8％±1.78％,명현고우50 μmol/L1,4-BQ염독조(35.9％±4.51％),이상각조적차이균유통계학의의(P＜0.05).DNA Ladder법결과여류식세포의검측수거기본일치.여NU7026조화1,4-BQ염독조비교,NU7026+1,4-BQ조Bax mRNA표체수평승고;여Wortmannin조화1,4-BQ조비교,Wortmannin+1,4-BQ조Bax mRNA표체수평승고,차이균유통계학의의(P＜0.05).Western blot검측HL60세포불표체p53단백.결론 DNA-PK억제제NU7026화Wortmannin촉진1,4-BQ유도적비p53의뢰적HL60세포조망.
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 significance (P＜0.05).However,p53 protein was not detected in HL60 cells as the western blot indicated.Conclusion Inhibitors of DNA-PK,NU7026 and Wortmannin,promote p53-independent apoptosis induced by 1,4-benzoquinone in HL60 cells.
