DNA-PKcs在石英诱导的DNA双链断裂修复中的作用  

Role of DNA-dependent protein kinase catalytic subunit in silica-induced DNA doublelstrand break repair in human embryo lung fibroblasts

作　　者：张凤梅[1] 刘秉慈[1] 刘海峰[1] 贾效伟[1] 叶萌[1] 

机构地区：[1]中国疾病预防控制中心职业卫生与中毒控制所,北京100050

出　　处：《中华劳动卫生职业病杂志》2009年第1期2-6,共5页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：国家自然科学基金项目（30671747,30371206）

摘　　要：目的探讨DNA依赖性蛋白激酶催化亚单位（DNA-dependent protein kinase catalytic subunit，DNA-PKcs）在石英致人胚肺成纤维细胞（HELF）DNA双链断裂修复中的作用。方法脂质体转染法建立DNA-PKcs的小干扰RNA（siRNA）及其阴性对照重组质粒稳定转染HELF系（简称HELF-PKcs和HELF-NC）。3种方式分组及对细胞的处理：（1）25、50、100、200、300、400μg／ml浓度的石英刺激HELF 12h；（2）200μg／ml的石英刺激HELF 0、1、2、6、12、24h；（3）200μg／ml的石英刺激HELF，PKcs和HELF-NC 0、12、24h。用免疫印迹法检测DNA-PKcs表达、磷酸化H2AX（H2AX）的水平，用Image-Proplus 6．0软件对条带光强度进行半定量分析；用中性彗星实验（彗尾DNA百分含量值）判断石英诱导的DNA双链断裂损伤强度。结果不同浓度的石英刺激HELF 12h，γH2AX水平及彗尾DNA百分含量随着石英浓度的增加逐渐升高。200μg／ml的石英刺激HELF 6h时，彗尾DNA百分含量[（38．7±6．9）％]与对照组相比明显增加，并且在12h达峰值，24h相对12h时点明显降低，差异均有统计学意义（P〈0．05）。在抑制DNA-PKcs的表达的细胞，12h时，石英刺激的HELF-PKcs的石英诱导的γH2AX水平增加受抑制，彗尾DNA百分含量为（43．09±3．68）％，与石英刺激的HELF-NC相比，差异无统计学意义（P〉0．05）；24h时，石英刺激的HELF-PKcs的石英诱导的γH2AX水平与石英刺激的HELF-NC相比无明显差异，差异无统计学意义（P〉0．05）。石英刺激的HELF-PKcs的彗尾DNA百分含量[（35．79±4．26）％]明显高于石英刺激的HELF-NC，差异有统计学意义（P〈0．05）。结论石英可诱导DNA双链断裂损伤，DNA-PKcs是石英诱导的DNA双链断裂损伤的感受器，通过磷酸化H2AX，促进石英诱导的DNA双链断裂损伤的修复。Objective To study the role of DNA-dependent protein kinase catalytic subunit （DNA- PKcs） in silica-induced DNA double-strand break repair in human emblyo lung fibroblasts （HELF）. Methods Two stable transfectants,HELF transfected with DNA-PKcs siRNA （HELF-PKcs） and with negative control siRNA （HELF-NC）, were established. HELF cells were treated with 0,25,50,100,200,300 and 400 μg/ml silica for 12 h and with 200 μg/ml silica for different times （0,1,2,6, 12 and 24 h ）. HELF-PKcs and HELF- NC were treated with 200 μg/ml silica for 0, 12 and 24 h. The expression levels of DNA-PKcs and phosphor- H2AX（H2AX） were determined by Western blot. DNA double strand breaks were measured by neutral comet assay. Results After treatment with different doses of silica for 12 h,the levels of H2AX and the percentages of tail DNA increased in concentration-dependent manner. After treatment with 200 μg/ml silica for different times,the levels of H2AX increased in a time-dependent manner. The percentages of tail DNA increased significantly at 6 b,and reacbing maximum at 12 h and then decreasing at 24 b. The expression level of DNA- PKcs was suppressed in HELF-PKes. After treatment with silica at 12 h,the level of H2AX was lower in HELF-PKcs than in HELF-NC,and the percentages of tail DNA increased obviously in both HELF-PKcs and HELF-NC compared with non-treated cells,but no significant difference was found in the percentages of tail DNA between them. The percentages of tail DNA decreased markedly in silica-treated HELF-NC and was significantly lower than in HELF-PKes at 24 h （P〈0.05）. Conclusion Silica can induce DNA double strand breaks in human embryo lung fibroblasts. DNA-PKcs might play a major role in siliea-induced DNA double strand break repair. Silica-induced histone H2AX phospborylation was dependent on DNA-PKcs.

关 键 词：蛋白激酶类 石英 DNA损伤 彗星试验 DNA修复 

分 类 号：R686[医药卫生—骨科学]