过氧化氢诱导HUVECs氧化应激模型的构建

Establishment of Oxidative Stress Model by Inducing Human Umbilical Vein Endothelial Cells Using Hydrogen Peroxide

目的利用过氧化氢(H2O2)诱导人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)构建体外氧化应激细胞模型.方法 H2O2分6个浓度梯度处理HUVECs不同时间,倒置显微镜观察细胞形态变化,CCK-8法检测细胞存活率,流式细胞术检测细胞凋亡率,DCFH-DA荧光探针和流式细胞仪检测细胞内活性氧水平,筛选H2O2的最佳作用浓度和时间.结果不同浓度H2O2处理细胞不同时间,对HUVECs均有损伤作用;400、600、800μmol/L H2O2处理HUVECs 24 h,细胞存活率显著降低(P<0.05),800μmol/L H2O2作用HUVECs不同时间,细胞存活率随处理时间延长逐渐降低(P<0.01);各组H2O2处理HUVECs 24 h,细胞凋亡率显著增加(1 000μmol/L除外,P<0.05),坏死率亦显著增加(100μmol/L除外,P<0.05);800μmol/L H2O2处理HUVECs不同时间,细胞凋亡率随处理时间延长逐渐增加,24 h时达到最高(P<0.05),细胞坏死率亦逐渐增加,48 h时达到最高(P<0.05);各组H2O2作用HUVECs 24 h,浓度400μmol/L时,细胞内ROS水平达到最高(P<0.01),800μmol/L H2O2处理HUVECs,胞内ROS水平随处理时间延长而递增(P<0.01).结论800μmol/L H2O2与HUVECs作用24 h可构建体外细胞氧化应激模型.

Objective To establish an oxidative stress cell model by inducing human umbilical vein endothelial cells（HUVECs） using hydrogen peroxide（H2 O2）. Methods Six gradient concentrations of H2 O2 were co-cultured with HUVECs for 6 h, 12 h, 24 h and 48 h. Inverted microscope was used to observe the change of cell morphologies. The optimal working concentration and effect time of H2 O2 on HUVECs were screened by CCK-8 method for cell viability, flowcymetry for apoptosis rate, and CDFU-DA fluorescent probing and flowcymetry for ROS levels. Re s ults Any concentration of H2 O2 could do harm to HUVECs co-culturing with cells. Cell viabilities decreased statistically when treating cells with 400, 600 or 800 μmol/L H2 O2 for different times（P0.05）. Cell viabilities also decreased gradually as time prolonged when treating cells with 800 μmol/L H2 O2（P0.001）. When treating cells with different concentration of H2 O2 for 24 h, cell apoptosis rates increased dramatically（1000μmol/L as an exception,P0.05）, so did cell necrosis rates（100 μmol/L as an exception,P0.05）;When treating cells with 800 μmol/L H2 O2, cell apoptosis rates increased dramatically as time prolonged, reaching the peak at 24 h（P0.05）, so did necrosis rates, reaching the peak at 48 h（P0.05）. ROS level reached thepeak while treating cells with 400 μmol/L H2 O2（P0.001）; ROS levels increased gradully as time prolonged when treating cells with 800 μmol/L H2 O2（P0.001）. Conclusions The oxidative stress model in HUVECs was established by coculturing cells with culture medium containing 800 μmol/L hydrogen peroxide for 24 h.