CAJ | 학술논문

目的探讨p53表达在石英致人胚肺成纤维细胞(HELF)细胞周期改变及DNA双链断裂(DNA double strand breaks,DSBs)修复中的作用.方法 3种方式分组处理细胞:(1)不同浓度(0、25、50、100、200、300、400μg/ml)的石英刺激HELF细胞12 h;(2)200 μg/ml石英刺激HELF细胞O、1、2,6、12、24 h;(3)200μg/ml石英刺激H-CMV和H-p53细胞O、12、24 h.用中性彗星试验检测石英所致的DSBs损伤强度,并计算DNA修复能力(DNA repair compentence,DRC),用免疫印迹法检测蛋白表达和磷酸化水平,用流式细胞仪检测细胞周期的变化.结果 (1)200μg/ml的石英作用HELF细胞不同时间,p53表达及p53-Ser15磷酸化水平随着作用时间的延长逐渐升高,12 h达峰值,24 h较12 h略有降低;不同浓度的石英作用HELF细胞12 h,随着石英浓度的增加,p53表达及p53-Ser15磷酸化水平逐渐增强,呈现剂量-反应关系.(2)石英作用于p53 siRNA的阴性对照细胞H-CMV,DRC为57.19%:石英作用沉默p53表达的H-p53细胞后,DSBs的修复能力增强,DRC为87.68%.(3)石英作用p53siRNA的阴性对照细胞24 h后,S期细胞比例由(24.3±3.8)%增加到(31.8±1.1)%,差异有统计学意义(P<0.05);沉默p53表达后,石英诱导的HELF细胞S期细胞比例[(41.4±0.6)%]与同期对照组[(25.4±1.9)%]相比有明显增加.差异有统计学意义(P<0.05).结论石英可诱导p53表达及磷酸化水平的增加,p53表达上调可抑制石英所致S期细胞百分比的增加及石英所致DNA双链断裂的修复.
목적 탐토p53표체재석영치인배폐성섬유세포(HELF)세포주기개변급DNA쌍련단렬(DNA double strand breaks,DSBs)수복중적작용.방법 3충방식분조처리세포:(1)불동농도(0、25、50、100、200、300、400μg/ml)적석영자격HELF세포12 h;(2)200 μg/ml석영자격HELF세포O、1、2,6、12、24 h;(3)200μg/ml석영자격H-CMV화H-p53세포O、12、24 h.용중성혜성시험검측석영소치적DSBs손상강도,병계산DNA수복능력(DNA repair compentence,DRC),용면역인적법검측단백표체화린산화수평,용류식세포의검측세포주기적변화.결과 (1)200μg/ml적석영작용HELF세포불동시간,p53표체급p53-Ser15린산화수평수착작용시간적연장축점승고,12 h체봉치,24 h교12 h략유강저;불동농도적석영작용HELF세포12 h,수착석영농도적증가,p53표체급p53-Ser15린산화수평축점증강,정현제량-반응관계.(2)석영작용우p53 siRNA적음성대조세포H-CMV,DRC위57.19%:석영작용침묵p53표체적H-p53세포후,DSBs적수복능력증강,DRC위87.68%.(3)석영작용p53siRNA적음성대조세포24 h후,S기세포비례유(24.3±3.8)%증가도(31.8±1.1)%,차이유통계학의의(P<0.05);침묵p53표체후,석영유도적HELF세포S기세포비례[(41.4±0.6)%]여동기대조조[(25.4±1.9)%]상비유명현증가.차이유통계학의의(P<0.05).결론 석영가유도p53표체급린산화수평적증가,p53표체상조가억제석영소치S기세포백분비적증가급석영소치DNA쌍련단렬적수복.
Objective To study the role of p53 in silica-induced cell cycle ahernation and DNA dou-ble strand breaks repair in human embryo lung fibroblasts(HELF).Methods Neutral comet assay was ap-plied to detect silica-induced DNA double strand breaks.According to the neutral comet experimental result,the DNA repair competence was calculated.The expression levels and phosphorylation of protein in HELF were determined by Western blot.Cell cycle changes were identified by flow cytometry in HELF.Resuits Af-ter treatment with 200 μg/ml silica for different times(0,1,2,6,12 and 24 h),the expression levels and Dhos.phorylation of p53 increased in a time-dependent manner,reaching maximum at 12 h and then decreasing at 24 h.After treatment with 0,25,50,100,200,300 and 400μg/ml silica for 12 h,the expression levels and phosphorylation of p53 lncreased in concentration-dependent manner.After p53 expression was inhibited,sili-ca-induced DNA damage repair competence was markedly increased(DRC=87.68%),compared with the negative control cell induced by silica(DRC=57.19%).Silica increased the percentage of S phase(31.8±1.1)%compared with the controls(24.3±3.8)%(p<0.05).When p53 expression was inhibited,the number of S phase cells was significantly increased,(41.4±0.6)%compared with the controls(25.4±1.9)%(P<0.05).Conclusion The silica dramatically increases the expression levels and phosphorylation of p53.The in-creased expression of p53 mediates silica-induced cell cycle change and inhibits silica-induced DNA double strand breaks repair.