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目的探讨过氧化氢(H2O2)诱导Dami细胞氧化应激模型的条件,建立并评价模型。方法不同浓度过氧化氢处理Dami细胞后,Giemsa染色,显微镜观察细胞形态学的变化;CCK-8试剂盒检测细胞增殖活性,绘制生长曲线,计算半数抑制浓度(IC50);加载DCFH-DA探针后流式细胞仪检测细胞内活性氧自由基水平;Western blot检测核因子NF-E2相关因子(Nrf2)、醌氧化还原酶(NQO1)、血红素加氧酶(HO-1)、γ-谷氨酰半胱氨酸合成酶催化亚单位(GCLC)表达量。结果随着H2O2浓度升高,Dami细胞核皱缩趋于明显,存活率下降,IC50为(0.9132±0.144)mmol/L;0.1 mmol/L、1 mmol/L、10 mmol/L H2O2处理Dami细胞2 h,细胞氧化应激阳性率分别为12.11%、20.91%、52.53%;H2O2处理Dami细胞24 h后,抗氧化蛋白Nrf2、GCLC水平升高,未检测到HO-1和NQO1的表达。结论 1 mmol/L H2O2是建立Dami细胞氧化应激模型的合适浓度。Dami细胞抗氧化应激反应与Nrf2/ARE通路有关。
Objective To investigate the conditions of hydrogen peroxide (H2O2) -induced oxidative stress in Dami cells and establish and evaluate the model. Methods Dami cells were treated with different concentrations of hydrogen peroxide and stained with Giemsa. The morphological changes of cells were observed by microscope. Cell proliferation was measured by CCK-8 kit and the growth curve was plotted to calculate the half maximal inhibitory concentration (IC50). DCFH-DA probe The levels of reactive oxygen species (ROS) in cells were detected by flow cytometry. The expressions of NF-E2, NQO1, HO-1, Cysteine synthase catalytic subunit (GCLC) expression level. Results With the increase of H2O2 concentration, the cell nucleus of Dami tended to shrink obviously, and the survival rate decreased. The IC50 was (0.9132 ± 0.144) mmol / L. Dami cells were treated with 0.1 mmol / L, 1 mmol / L and 10 mmol / h. The positive rates of oxidative stress were 12.11%, 20.91% and 52.53%, respectively. After treated with H2O2 for 24 h, the levels of Nrf2 and GCLC were increased, and the expressions of HO-1 and NQO1 were not detected. Conclusion 1 mmol / L H2O2 is the suitable concentration for establishing oxidative stress model in Dami cells. Dami cell antioxidant stress response and Nrf2 / ARE pathway.