磷脂酰肌醇-3激酶对石英诱导的细胞周期改变及Ku80蛋白表达的影响

Effects of phosphatidylinositol 3 kinase on silica-induced cell cycle changes and expression of Ku80

目的探讨磷脂酰肌醇-3激酶(Phosphatidylinositol 3 kinase,PI3K)是否通过对Ku80蛋白水平的调节,参与石英诱导的细胞周期改变,为石英粉尘的防治工作提供理论依据。方法采用显性失活突变体技术抑制PI3K功能,RNAi技术抑制Ku80蛋白表达,流式细胞技术检测细胞周期,免疫印迹技术检测Ku80蛋白的表达,并用Image-Proplus 6.0软件对条带光强度进行半定量分析。结果石英刺激人胚肺成纤维细胞(human embryonic lung fibroblast,HELF)对照组,G1期细胞所占比例从(83.53±2.24)%下降到(69.11±3.12)%,差异有统计学意义(P<0.05);石英刺激抑制PI3K功能的DN-△p85细胞组,G1期细胞所占比例下降趋势减弱,从(82.16±2.43)%仅下降为(79.39±2.35)%,差异无统计学意义;石英刺激抑制Ku80蛋白表达的H-Ku80细胞组,G1期细胞所占比例进一步减少,从(85.16±3.73)%下降到(59.92±3.31)%,差异有统计学意义(P<0.05);抑制PI3K功能后,石英引起的Ku80蛋白水平增高受抑制。结论 PI3K对石英诱导的细胞周期改变有正性调控作用,该调控过程是Ku80蛋白非依赖性的。

Objective To study whether phosphatidylinositol 3 kinase （PI3K） participated in silica -induced cell cycle changes by adjusting the protein level of Ku80, so as to provide basis for the control and prevention work of quartz dust. Methods The dominant inactivation mutant technology was used to restrain the function of PI3 K, and the RNAi technology was adopted to restrain the protein expression of Ku80. We used flow cytome- try to detect the cell cycle and immunobloting technique to detect the protein expression of Ku80. At the same time, strip light intensity for semi - quantitative analysis was conducted by Image - Pro plus 6.0 software. Re- sults Quartz can stimulate human embryonic lung fibroblast （HELF） . The proportion of HELF in G1 phases decreased from （83.53±2.24）% to （69. 11 ±3.12 ）%, and there was statistical significant difference （P 〈 0. 05 ） . Quartzb stimulation restrained PI3K function of DN - Ap85 cell group and the decline trend of per- centage of G1 cells weakened which decreased from （ 82. 16± 2.43 ） % to （79.39 ± 2. 35 ） % and there was no statistical significant difference. Quartzb stimulation restrained the protein expression of Ku80 of H - Ku80 cell group and the percentage of G1 cells group further decreased, which decreased from （ 85.16 ± 3.73 ） % to （59.92± 3.31 ）% and there was statistical significant difference. After inhibition of PI3K function, the in- crease of protein level of Ku80 caused by quartz was obviously suppressed. Conclusions PI3 K had positive adjusting effects on cell cycle changes and the process was independent.