过氧化氢诱导人卵巢颗粒细胞氧化应激模型的建立与评价  

Establishment and evaluation of a model of hydrogen peroxide-induced oxidative stress in human ovarian granulosa cells

作　　者：徐强 张曼丽 腊晓琳[1] Xu Qiang;Zhang Manli;La Xiaolin(Center for Reproductive Medicine,First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,China)

机构地区：[1]新疆医科大学第一附属医院生殖医学中心,乌鲁木齐830054

出　　处：《中华生殖与避孕杂志》2025年第2期172-182,共11页Chinese Journal of Reproduction and Contraception

基　　金：中央引导地方科技发展资金项目(ZYYD2024ZY07);国家自然科学基金(81960289);自治区研究生创新项目(XJ2024G152)。

摘　　要：目的探讨利用过氧化氢(H_(2)O_(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_(2)O_(2)组,对照组不作处理,H_(2)O_(2)组分别给予200μmol/L、400μmol/L、600μmol/L、800μmol/L、1000μmol/L H_(2)O_(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_(2)O_(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_(2)O_(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_(2)O_(2)浓度200μmol/L、400μmol/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_(2)O_(2)浓度增加至600μmol/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_(2)O_(2)浓度继续增加至800μmol/L、1000μmol/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_(2)O_(2)浓度分别为200μmol/L、400μmol/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_(2)O_(2)浓度分别增加至600μmol/L、800μmol/L、1000μmol/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_(2)O_(2)浓度600μmol/L时P=0.011,800μmol/L时P=0.003,1000μmol/L时P=0.005;相对ROS强度:H_(2)O_(2)浓度600μmol/L时P=0.002,800μmol/L及1000μmol/L时P<0.001)。与对照组相比,使用不同浓度的H_(2)O_(2)干预后,细胞线粒体膜电位逐渐ObjectiveTo establish an oxidative stress injury model by using hydrogen peroxide(H_(2)O_(2))to induce human ovarian granulosa cells COV434.MethodsHuman ovarian granulosa cells line COV434 were randomly divided into 6 groups,control group was not treated,H_(2)O_(2) groups were treated with H_(2)O_(2) of 200μmol/L,400μmol/L,600μmol/L,800μmol/L and 1000μmol/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_(2)O_(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_(2)O_(2) for intervention,and the ROS level and mitochondrial membrane potential were measured.ResultsThe cell viability of the 200μmol/L and 400μmol/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_(2)O_(2) increased to 600μmol/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_(2)O_(2) continued to increase to 800μmol/L and 1000μmol/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_(2)O_(2) was 200μmol/L and 400μmol/L,there was no significant difference

关 键 词：颗粒细胞 氧化应激损伤 细胞模型 过氧化氢 卵巢储备功能减退 

分 类 号：R711.75[医药卫生—妇产科学]