p53表达在石英诱导的细胞周期改变及DNA损伤修复中的作用

摘要

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.%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.