过氧化氢诱导人肺动脉内皮细胞损伤模型的建立及其氧化应激机制  

Establishment of a model of hydrogen peroxide-induced injury in pulmonary artery endothelium cells and relevant mechanisms of oxidative stress

作　　者：叶珏[1] 何阳阳[1] 颜艺[2] 赵隽涵 连天宇 王晓建[1] 阎雨[3] 张思瑾[2] 杨淑慧[1] 荆志成[1] 

机构地区：[1]中国医学科学院北京协和医学院国家心血管病中心阜外医院心血管疾病国家重点实验室,100037 [2]同济大学附属上海市肺科医院肺循环科 [3]中国医学科学院北京协和医学院药物研究所天然药物活性物质与功能国家重点实验室

出　　处：《中华心血管病杂志》2017年第7期613-618,共6页Chinese Journal of Cardiology

基　　金：国家自然科学基金（81320108005,81630003）;国家杰出青年科学基金（81425002）;中国医学科学院医学与健康科技创新工程（2016-I2M-1-002）

摘　　要：目的 建立过氧化氢（H2O2）诱导的人肺动脉内皮细胞（PAEC）损伤模型,探讨氧化应激促使PAEC结构和功能改变的分子机制.方法 采用不同浓度（25、50、100、200、400、800、1 600、3 200和6 400 μmol/L）的H2O2刺激PAEC,分别作用4、24 h后,采用CCK-8法测定PAEC活力并绘制存活曲线,采用流式细胞术检测PAEC凋亡情况,利用小分子荧光探针检测PAEC内活性氧（ROS）生成和线粒体活性,采用Western blot法检测PAEC内信号分子的磷酸化水平.结果 （1）不同浓度H2O2对PAEC活力的影响：H2O2作用4h后,PAEC的活力随着H2O2浓度的增加而降低.同时,经过计算H2O2作用4和24h后,H2O2抑制PAEC活力的IC50值分别为397.00和488.77 μmol/L.（2）H2O2对PAEC凋亡的影响：400 μmol/L的H2O2作用4h后,PAEC晚期凋亡和坏死的比率分别为（22.58 ±3.69）％和（11.86±4.27）％,而阴性对照PAEC晚期凋亡和坏死的比率仅为（3.41±1.44）％和（1.94±1.15）％,P均〈0.05.H2O2处理后PAEC存活率仅为（7.98±3.21）％,明显低于阴性对照（48.89±8.08）％,P〈0.01.但H2O2处理后PAEC与阴性对照PAEC的早期凋亡率差异并无统计学意义.（3）H2O2对PAEC内线粒体活性和ROS生成的影响：H2O2处理后PAEC ROS生成量和线粒体活性均明显高于阴性对照（P均〈0.01）.（4） H2O2对PAEC内信号传递分子蛋白含量的影响：H2O2作用后30 min,PAEC内Akt含量与阴性对照PAEC比较差异无统计学意义,而p-Akt含量则明显高于阴性对照PAEC（P 〈0.01）,同时p-JNK和p-p38含量亦均明显高于阴性对照PAEC（P均〈0.01）.结论 400μmol/L H2O2可明显损伤人PAEC,其机制可能涉及Akt、MAPK等多条信号通路.Objective To establish a hydrogen peroxide （H2O2） induced injury model of pulmonary artery endothelial cells （PAECs） and explore the molecular mechanisms of oxidative stress on the structure and function of PAECs in this model.Methods Human PAECs were treated with H2O2 at different concentrations （25,50,100,200,400,800,1 600,3 200,6 400 μmol/L） for 4 and 24 h,respectively.The PAECs survival curve was obtained according to the cell viability measured by CCK-8 assay.The cell apoptosis of PAECs was detected by flow cytometry.The reactive oxygen species （ROS） generation and mitochondrial activity were measured using small molecule fluorescent probes.Proteins were extracted and the phosphorylation levels of signal molecules in PAECs were detected by Western blot assays.Results （1） The effect of H2O2 at various concentrations on cell viability of PAECs：cell viability of PAECs decreased in proportion to increasing concentration of H2O2 after incubation for 4 h.The half maximal inhibitory concentration （ICs0） of PAECs exposed to H2O2 for 4 and 24 h were 397.00 and 488.77 μmol/L,respectively.（2） The effect of H2O2 on cell apoptosis of PAECs：After H2O2 incubation for 4 h,proportions of PAECs at late-apoptosis （（22.58 ± 3.69） %） and necrotic stage（（11.86 ± 4.27）%） were significantly higher than those of control PAECs at late-apoptosis stage（（3.41 ± 1.44） %,P 〈 0.01） and at necrotic stage （（1.94 ± 1.15） %,P 〈0.05）.The survival rate of PAECs post H2O2 was dramatically lower than that of control PAECs （（7.98 ± 3.21） % vs.（48.89 ± 8.08） %,P 〈 0.01）.However,there is no statistical difference between both groups regarding to the early apoptosis.（3） The effect of H2O2 on mitochondrial activity and ROS production of PAECs：the mitochondrial activity and ROS generation of PAECs treated by H2O2 were significantly increased compared to those in control PAECs （P 〈0.01）.（4）The effect of H2 O2 on signaling molecules in PAECs：there w

关 键 词：肺动脉 内皮细胞 氧化性应激 过氧化氢 

分 类 号：R543[医药卫生—心血管疾病]