摘要目的:探讨利用过氧化氢（H 2O 2）诱导人卵巢颗粒细胞COV434建立氧化应激损伤模型的方法。 方法:将人卵巢颗粒细胞COV434细胞分为对照组及H 2O 2组，对照组不作处理，H 2O 2组分别给予200 μmol/L、400 μmol/L、600 μmol/L、800 μmol/L、1 000 μmol/L H 2O 2进行处理0.5 h、1 h、2 h、4 h、6 h，CCK-8法测定细胞活性，后续实验使用不同浓度H 2O 2处理1 h；β-半乳糖苷酶染色法测定细胞衰老程度；流式细胞术测定DCFH-DA荧光染色，测定细胞活性氧（reactive oxygen species，ROS）水平；JC-1染色法测定细胞线粒体膜电位；Western blotting法测定细胞凋亡相关蛋白Caspase-3、Caspase-9的表达水平。建立模型成功后，为验证细胞模型的可用性，使用抗氧化剂维生素E对细胞预处理12 h，随后加入H 2O 2进行干预，并对ROS水平、线粒体膜电位进行测定。 结果:与对照组相比，H 2O 2浓度200 μmol/L、400 μmol/L组的细胞存活率呈现先下降后上升的趋势，且在干预1 h后趋于稳定，各时间点细胞存活率差异均无统计学意义（均 P>0.05）；当H 2O 2浓度增加至600 μmol/L时，细胞存活率随处理时间逐渐下降并在1 h后趋于稳定，显著降低且最接近50%（ P<0.001）；当H 2O 2浓度继续增加至800 μmol/L、1 000 μmol/L时，细胞存活率随处理时间逐渐下降并在1 h后趋于稳定，且降至10%以下（均 P<0.001）。当H 2O 2浓度分别为200 μmol/L、400 μmol/L时，处理1 h后的β-半乳糖苷酶阳性细胞比值、相对ROS强度与对照组相比差异均无统计学意义（均 P>0.05）；当H 2O 2浓度分别增加至600 μmol/L、800 μmol/L、1 000 μmol/L时，β-半乳糖苷酶染色阳性细胞比值、相对ROS强度均显著增高（β-半乳糖苷酶染色：H 2O 2浓度600 μmol/L时 P=0.011，800 μmol/L时 P=0.003，1 000 μmol/L时 P=0.005；相对ROS强度：H 2O 2浓度600 μmol/L时 P=0.002，800 μmol/L及1 000 μmol/L时 P<0.001）。与对照组相比，使用不同浓度的H 2O 2干预后，细胞线粒体膜电位逐渐降低，且与H 2O 2浓度呈负相关（均 P<0.001）。与对照组相比，200 μmol/L H 2O 2组的Cleaved-Caspase-3表达差异无统计学意义（ P>0.05），在400 μmol/L、600 μmol/L、800 μmol/L、1 000 μmol/L H 2O 2组表达明显增加（均 P<0.001）。Caspase-3、Caspase-9在所有H 2O 2处理组中的表达均明显升高（均 P<0.001）。使用维生素E处理后，与模型组相比，维生素E组相对ROS强度显著降低（ P=0.009），细胞线粒体膜电位显著升高（ P<0.001），但尚不能恢复至对照组水平。 结论:使用600 μmol/L的H 2O 2持续处理COV434细胞1 h，可快速建立稳定有效的人卵巢颗粒细胞氧化应激损伤模型。

abstractsObjective:To establish an oxidative stress injury model by using hydrogen peroxide (H 2O 2) to induce human ovarian granulosa cells COV434. Methods:Human ovarian granulosa cells line COV434 were randomly divided into 6 groups, control group was not treated, H 2O 2 groups were treated with H 2O 2 of 200 μmol/L, 400 μmol/L, 600 μmol/L, 800 μmol/L and 1 000 μmol/L for 0.5 h, 1 h, 2 h, 4 h and 6 h, respectively, and the cell viability was determined by CCK-8 method. The follow-up experiments were treated with different concentrations of H 2O 2 for 1 h. β-galactosidase staining was used to determine the degree of cell senescence. DCFH-DA fluorescence staining was determined by flow cytometry, and the level of reactive oxygen species (ROS) in cells was determined. JC-1 staining was used to determine the mitochondrial membrane potential of cells. Western blotting was used to determine the expression levels of apoptosis-related proteins Caspase-3 and Caspase-9. After the successful establishment of the model, in order to verify the usability of the cell model, the cells were pretreated with the antioxidant vitamin E for 12 h, followed by the addition of H 2O 2 for intervention, and the ROS level and mitochondrial membrane potential were measured. Results:The cell viability of the 200 μmol/L and 400 μmol/L groups decreased first and then increased compared with the control, and tended to be stable after 1 h of intervention, and there was no significant difference in cell viability at each time point (all P>0.05). When the concentration of H 2O 2 increased to 600 μmol/L, the cell viability gradually decreased with the treatment time and tended to stabilize after 1 h, and decreased significantly to nearly 50% ( P<0.001). When the concentration of H 2O 2 continued to increase to 800 μmol/L and 1 000 μmol/L, the cell viability gradually decreased with the treatment time and stabilized after 1 h, and decreased to less than 10% (all P<0.001). When the concentration of H 2O 2 was 200 μmol/L and 400 μmol/L, there was no significant difference in the ratio of β-galactosidase-positive cells and the relative ROS intensity after 1 h compared with the control (all P>0.05). When the concentration of H 2O 2 increased to 600 μmol/L, 800 μmol/L and 1 000 μmol/L, the ratio of β-galactosidase-positive cells and the relative ROS intensity increased significantly (β-galactosidase staining: P=0.011 at 600 μmol/L, P=0.003 at 800 μmol/L, P=0.005 at 1 000 μmol/L; the relative ROS intensity: P=0.002 at 600 μmol/L, P<0.001 at 800 μmol/L and 1 000 μmol/L). Compared with the control, the mitochondrial membrane potential of cells decreased gradually after intervention with different concentrations of H 2O 2, and was negatively correlated with H 2O 2 concentration (all P<0.001). There was no difference in the expression of Cleaved-Caspase-3 in the H 2O 2 group at 200 μmol/L compared with the control, and the expression was significantly increased at 400 μmol/L, 600 μmol/L, 800 μmol/L and 1 000 μmol/L (all P<0.001). The expressions of Caspase-3 and Caspase-9 were significantly increased in all H 2O 2 treated groups (all P<0.001). Compared with the model group, the relative ROS intensity of the vitamin E group was significantly reduced ( P=0.009), and the mitochondrial membrane potential was significantly increased ( P<0.001), but it could not be restored to the level of the control. Conclusion:Using 600 μmol/L H 2O 2 to continuously treat COV434 cells for 1 h can quickly establish a stable and effective oxidative stress injury model of human ovarian granulosa cells.