磷脂酰肌醇-3激酶在石英诱导的DNA双链断裂修复中的作用

目的 探讨磷脂酰肌醇-3激酶(phosphatidylinositol 3 kinase,PI3K)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 用200μg/ml的石英刺激HELF和用显性失活突变体抑制P13K功能的HELF(DN-Δp85)不同时间.免疫印迹法检测磷酸化H2AX(γH2AX)的水平以及DNA依赖性蛋白激酶(DNA-dependent protein kinase,DNA-PK)的组成成分Ku70、Ku8和DNA-PKcs的蛋白水平,并用Image-Pro plus 6.0软件对条带光强度进行半定量分析.中性彗星试验检测DNA双链断裂损伤,用彗尾DNA百分含量值观察DNA双链断裂损伤程度变化,并计算DNA修复能力.结果 γH2AX的水平在石英刺激3 h明显增高,12 h达峰值,24 h下降.与HELF相比,石英诱导的DN-Δp85细胞γH2AX水平增高受到抑制.石英刺激HELF和DN-ΔP85细胞12 h组Ku70、Ku80和DNA-PKcs的蛋白水平(0.58±0.09、0.95±0.21、0.55±0.06,0.37±0.14、0.55±0.17、0.52±0.07)均高于相应的无石英刺激组(0.26±0.10、0.69±0.26、0.43±0.11,0.11±0.07、0.27±0.14、0.39±0.07),差异有统计学意义(P<0.05).与石英刺激HELF 12 h组比较,石英刺激DN-ΔP85 12 h组的Ku70、Ku80蛋白水平增高受到抑制,差异均有统计学意义(P<0.05).石英刺激的HELF12 h组和DN-Δp85 12h组的彗尾DNA百分含量分别为9.78±1.15和11.79±4.90,明显高于同细胞系的无石英刺激组(2.40±0.69,3.31±1.35),差异有统计学意义(P<0.05);与石英刺激HELF 12 h组相比,石英刺激HELF细胞24 h组彗尾DNA百分含量明显降低(4.19±0.47),差异有统计学意义(P<0.05).石英刺激DN-Δp85 24 h组的彗尾DNA百分含量为(7.58±4.32),明显高于无石英刺激DN-Δp85组和石英刺激HELF 24 h组,差异有统计学意义(P<0.05).HELF的DNA修复能力为75.74%,DN-Δp85的DNA修复能力为49.64%.结论 石英可诱导DNA双链断裂损伤,PI3K与DNA损伤修复有关,通过调节Ku70和Ku80的水平,可促进石英诱导的DNA双链断裂损伤的修复.

Roles of phosphatidylinositol 3 kinase in silica-induced DNA double strand breaks damage repair in human embryo lung fibroblasts

Objective To study the role of Phosphatidylinositol 3 kinase(PI3K)in silica-induced DNA double strand break repair in human embryo lung fibroblasts(HELF).Methods Control HELF cells and DN-Δp85(HELF transfected with Dominant negative mutant of PI3K)were treated with 200μg/ml, silica for different times.The expression levels of phosphor-H2AX(H2AX),Ku70,Ku80 and DNA-PKcs were de-termined by Western blot.Furthermore,DNA double strand breaks were measuled by neutral comet assay after cells were treated with 200 μg/ml silica for 0,12 and 24 h.Results After treatment with 200 μg/ml silica for different times,the levels of H2AX were increased in a time-dependent manner and the expression levels of H2AX were obviously suppressed in DN-Δp85 compared with control cells.The levels of Ku70 and Ku80were also significantly suppressed in DN-Δp85(0.37±20.14,0.55±0.17)compared with control cells(0.58±0.09,0.95±0.21)after treatment with 200 μg/ml silica for 12 h(P<0.05).Both the percentage of tail DNA in HELF and DN-Δp85 increased significantly at 12 h(9.78±1.15,11.79±4.90)compared with groups without treatment with silica(2.40±0.69,3.31±1.35)and then decreased at 24 h(4.19±0.47,7.58±4.32),but only the decrease of HELF at 24 h was significant compared with HELF at 12 h(P<0.05).DNA repair competence of HELF was 75.74%and that of DN-Δp85 declined to 49.64%.Conclusion Silica dust can induce DNA double strand breaks in human embryo lung fibroblasts.P13K might play a role in silica-induced DNA double strand break repair by regulating the expression levels of Ku70 and Ku80.