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目的探讨Ku80蛋白在石英诱导的人胚肺成纤维细胞(HELF)细胞周期改变中的作用及机制。方法200μg/m L石英按0、3、6、12、24 h处理各组细胞,采用RNAi技术抑制Ku80蛋白表达(H-Ku80),采用流式细胞术检测石英刺激24 h不同细胞系的细胞周期变化情况,免疫印迹技术检测不同细胞系各时间点细胞周期蛋白D1(cyclin D1)、细胞周期蛋白依赖激酶4(CDK4)、E2增强子连接因子1(E2F1)蛋白表达和视网膜母细胞瘤蛋白(Rb)表达及Rb-ser780磷酸化水平。结果石英刺激对照细胞,G1期细胞所占比例从(89.28±2.19)%下降到(68.93±3.79)%;石英刺激H-Ku80,G1期细胞比例进一步减少,从(85.16±3.73)%下降到(59.92±3.31)%,差异均有统计学意义(P<0.05);抑制Ku80蛋白表达后,Rb蛋白表达水平无变化,石英诱导的CyclinD1、CDK4水平增高受抑制,而E2F1、Rb-ser780磷酸化水平进一步增高。结论 Ku80参与石英诱导的细胞周期改变,对CyclinD1、CDK4的表达呈正性调节作用;而对E2F1表达及Rb-ser780磷酸化呈负性调节作用。
Objective To investigate the role and mechanism of Ku80 protein in quartz-induced human embryonic lung fibroblasts (HELF) cell cycle changes. Methods The cells were treated with 200 μg / mL quartz for 0, 3, 6, 12 and 24 h. The expression of Ku80 protein was inhibited by RNAi (H-Ku80). The cells in different cell lines stimulated by quartz for 24 h were detected by flow cytometry Western blotting was used to detect the expression of cyclin D1, CDK4, E2F1 protein and retinoblastoma protein in different cell lines at different time points (Rb) expression and Rb-ser780 phosphorylation level. Results The percentage of cells in G1 phase decreased from (89.28 ± 2.19)% to (68.93 ± 3.79)% in Quartz-stimulated control cells. The percentage of cells in G1 phase decreased further from (85.16 ± 3.73)% to (59.92 ± 3.31)% respectively, the differences were statistically significant (P <0.05). The expression of Rb protein did not change after inhibiting the expression of Ku80 protein, the increase of CyclinD1 and CDK4 induced by quartz was inhibited, while the expression of E2F1, Rb-ser780 The level of further increase. Conclusion Ku80 participates in quartz-induced changes of cell cycle, which positively regulates the expressions of CyclinD1 and CDK4, while negatively regulates E2F1 expression and Rb-ser780 phosphorylation.