Objective To investigate the expressions of phosphorylated H2AX (γH2AX) and p53-binding protein 1 (53BP1) in DNA oxidative damage of human bronchial epithelial (HBE) cells.Methods The HBE cells were treated with 0,25,50,100,200,400 μmol/L of hydrogen peroxide (H2O2) for 1 hour,respectively,and their DNA oxidative damages displaying as double-strand breaks (DSBs) were induced.The viability and apoptosis of HBE cells were measured by the CCK-8 method and flow cytometry,respectively.The expression status of γH2AX and 53BP1 in nucleus of HBE cells was observed by a fluorescence microscope.The expression levels of γH2AX,53BP1 and BRCA1 were determined by western blot.Results Compared with the control (0 μmol/L of H2O2),the via bility of HBE cells treated with 25 μmol/L of H2O2 (1.07 ±0.01) increased,while those with 50,100,200,400 μmol/L of H2O2 (0.97 ± 0.01,0.96 ± 0.01,0.95 ± 0.01,0.94 ± 0.01) decreased significantly (F =50.35,P ＜ 0.01).The apoptosis rates of HBE cells treated with 50,100,200,400 μ mol/L of H2O2 ([7.54 ± 0.57] ％,[7.84 ± 0.68] ％,[8.40 ± 0.50] ％ and [14.03 ± 1.03] ％) were significantly higher than that with 0 μmol/L of H2O2 ([4.65 ± 0.32] ％,F =35.879,P ＜ 0.01).Compared with the control (0 μmol/L of H2O2),the average fluorescence intensity of γH2AX in nucleus of HBE cells treated with 25,50,100,200,400 μmol/L of H2O2 increased significantly (F =223.97,P ＜ 0.01),while those of 53BP1 in nucleus of HBE cells treated with 50,100,200,400 μmol/L of H2O2 decreased significantly (F =117.78,P ＜ 0.01).The results of western blot showed that the expres sion levels of γH2AX increased with the increase of H2O2 concentration,while that of 53BP1 and BRCA1 was on the contrary (F =96.20,21.92 and 11.55,respectively,P ＜0.01).Conclusion In the oxidative damage of HBE cells induced by H2O2,γH2AX may be used as a marker of DNA oxidative damage,while the decreased expression of 53BP1 suggests that other mechanisms to repair the DNA damage sites may exist.%目的 探讨肺正常上皮细胞(human bronchial epithelial cells,HBE)中磷酸化组蛋白H2AX(phosphorylated H2AX,γH2AX)和p53结合蛋白1(p53-Binding protein 1,53BP1)在DNA氧化损伤反应中的表达.方法 用0、25、50、100、200、400μμmol/L过氧化氢(H2O2)处理HBE细胞1h,对其造成氧化损伤引起DNA双链断裂(double strand breaks,DSBs);CCK-8比色法检测H2O2对HBE细胞活性的影响,流式细胞术检测细胞凋亡情况,荧光显微镜检测HBE细胞核内γH2AX和53BP1蛋白的聚集情况,western blot检测γH2AX、53BP1、BRCA1蛋白的表达水平.结果 与对照组比较,25 μmol/L H2O2处理组细胞活性(1.07±0.01)升高,50、100、200、400 μmol/L H2O2处理组细胞活性(分别为0.97±0.01,0.96 ±0.01,0.95±0.01和0.94±0.01)降低,差异具有统计学意义(F=50.35,P＜0.01).细胞凋亡检测结果显示,与对照组[(4.65±0.32)％]比较,50、100、200、400μmol/L H2O2处理组细胞凋亡率[分别为(7.54±0.57)％、(7.84±0.68)％、(8.40±0.50)％和(14.03±1.03)％]升高(F=35.879,P＜0.01).与对照组比较,经25、50、100、200、400 μmol/L H2O2处理后γH2AX的平均荧光强度升高(F=223.97,P＜0.01),而经50、100、200、400 μmol/L H2O2处理后53BP1的平均荧光强度降低(F=117.78,P＜0.01);western blot结果显示,随着H2O2浓度升高,γH2AX蛋白表达水平升高,BRCA1和53BP1的表达水平下降,差异均具有统计学意义(F分别为96.20,11.55,21.92,P均＜0.01).结论 在H2O2导致HBE细胞的氧化损伤中,γH2AX可作为DNA氧化损伤标志物,但53BP1的表达下降提示HBE细胞可能通过其他途径进行DNA氧化损伤的修复.
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