CAJ | 학술논문

目的探讨DNA依赖性蛋白激酶(DNA dependent protein kinase,DNA-PK)在石英诱导的人胚肺成纤维细胞(HELF)中细胞周期蛋白E(CyclinE)、细胞周期蛋白依赖激酶2(CDK2)蛋白表达及细胞周期改变中的作用.方法 200μg/ml石英处理阴性对照细胞(H-NC)及采用RNAi技术分别抑制DNA-PKcs和Ku80蛋白表达的细胞系(H-PKcs和H-Ku80)0、3、6、12、24 h,采用流式细胞术检测石英刺激24 h不同细胞系的细胞周期变化情况,免疫印迹(Western blot)技术检测不同细胞系各时间点CyclinE、CDK2蛋白的表达水平,并用Image-Pro plus 6.0软件对条带光强度进行半定量分析.结果石英刺激H-NC,G1期细胞所占比例从83.53%±2.24%下降到69.11%±3.12%;石英刺激H-Ku80,石英诱导的G1期细胞的比例进一步减少,从85.16%±3.73%下降到59.92%±3.31%;石英刺激H-PKcs,G1期细胞比例也显示进一步减少,从75.06%±2.23%下降到58.32%±1.35%,差异均有统计学意义(P＜0.05).石英刺激H-NC,CyclinE和CDK2蛋白表达随时间延长有增高趋势,并在12 h达峰值;石英刺激H-Ku80,H-PKcs和CyclinE蛋白表达水平在各时间点均低于H-NC,差异有统计学意义(P＜0.05),CDK2蛋白的表达水平变化不明显.结论 DNA-PK参与石英诱导的细胞周期改变,对CyclinE的表达呈正性调节作用.
목적 탐토DNA의뢰성단백격매(DNA dependent protein kinase,DNA-PK)재석영유도적인배폐성섬유세포(HELF)중세포주기단백E(CyclinE)、세포주기단백의뢰격매2(CDK2)단백표체급세포주기개변중적작용.방법 200μg/ml석영처리음성대조세포(H-NC)급채용RNAi기술분별억제DNA-PKcs화Ku80단백표체적세포계(H-PKcs화H-Ku80)0、3、6、12、24 h,채용류식세포술검측석영자격24 h불동세포계적세포주기변화정황,면역인적(Western blot)기술검측불동세포계각시간점CyclinE、CDK2단백적표체수평,병용Image-Pro plus 6.0연건대조대광강도진행반정량분석.결과 석영자격H-NC,G1기세포소점비례종83.53%±2.24%하강도69.11%±3.12%;석영자격H-Ku80,석영유도적G1기세포적비례진일보감소,종85.16%±3.73%하강도59.92%±3.31%;석영자격H-PKcs,G1기세포비례야현시진일보감소,종75.06%±2.23%하강도58.32%±1.35%,차이균유통계학의의(P＜0.05).석영자격H-NC,CyclinE화CDK2단백표체수시간연장유증고추세,병재12 h체봉치;석영자격H-Ku80,H-PKcs화CyclinE단백표체수평재각시간점균저우H-NC,차이유통계학의의(P＜0.05),CDK2단백적표체수평변화불명현.결론 DNA-PK삼여석영유도적세포주기개변,대CyclinE적표체정정성조절작용.
Objective To study the roles of DNA dependent protein kinase (DNA-PK)in silicainduced cell cycle changes and expressions of CyclinE and CDK2 in human embryo lung fibroblasts (HELF).Methods The expressions of Ku80 and DNA-PKcs proteins were inhibited by siRNA plasmids, respectively.Flow cytometry was used to detect the distributions of cell cycle and western blot assay was used to determine the expression levels of CyclinE and CDK2 after cells were exposed to 200 μg/ml silica for 0,3,6,12,24 h.Results The proportion of G1 phases in negative control cells decreased from 83.53%±2.24% to 69.11%±3.12%after exposure to silica; the proportion of G1 phases in H-Ku80 and H-PKcs cells exposed to silica decreased from 85.16%±3.73% to 59.92%±3.31% and from 75.06%±2.23% to 58.32%±1.35%, respectively (P＜0.05).The exposure to silica resulted in the increasing protein expression levels of CyclinE and CDK2 in negative control cells, and the expression levels of CyclinE were obviously suppressed in H-Ku80 and H-PKcs as compared with control cells. However, the expression level of CDK2 protein did not change significantly.Conclusion DNA-PK might play a role in silica-induced alternations of cell cycle and regulate silica-induced overexpression of CyclinE in human embryo lung fibroblasts.