硝酸甘油联合β-巯基乙醇对冠心病患者外周血内皮祖细胞的影响  

Influence of coadministration of β-mercaptoethanol and nitroglycerin on cell viability of peripheral blood-derived endothelial progenitor cells in patients with coronary heart disease

作　　者：曾彩雨 贺红[2] 杨发林[3] 潘建[1] 宁建文[1] 刘鑫 ZENG Cai-yu;HE Hong;YANG Fa-lin;PAN Jian;NING Jian-wen;LIU Xin(Department of Emergency Medicine,The First Affiliated Hospital,College of Medicine,Zhejiang University,Hangzhou 310003,China;Department of Cardiology,Qilu Hospital,Shandong University,Jinan 250012,China;Department of Clinical Laboratory,Qilu Hospital,Shandong University,Jinan 250012,China)

机构地区：[1]浙江大学医学院附属第一医院急诊科,浙江杭州310003 [2]山东大学齐鲁医院心内科,山东济南250012 [3]山东大学齐鲁医院检验科,山东济南250012

出　　处：《中国病理生理杂志》2018年第8期1427-1433,共7页Chinese Journal of Pathophysiology

摘　　要：目的:观察硝酸甘油对冠心病患者外周血内皮祖细胞(EPCs)体外增殖的影响及其分子机制,并探讨β-巯基乙醇改善硝酸甘油耐药的作用途径。方法:选择冠心病患者75例,分为硝酸甘油应用组和对照组,流式细胞术检测外周血中EPCs的数量,ELISA法检测血浆中血管内皮生长因子A(vascular endothelial growth factor-A,VEGF-A)和过氧亚硝基阴离子(ONOO^-)的水平;EPCs培养实验中分别用不同浓度硝酸甘油及β-巯基乙醇进行干预,MTT法检测EPCs的活力,ELISA法检测上清液VEGF-A和ONOO^-的含量,DCFH-DA探针检测细胞活性氧水平,Western blot检测贴壁细胞Akt、p-Akt、内皮型一氧化氮合酶(e NOS)和p-e NOS的蛋白水平。结果:与对照组相比,高浓度硝酸甘油应用组外周血EPCs显著减少,血清中ONOO^-增多,VEGF-A减少。适量浓度硝酸甘油组EPCs的活力提高,上清液VEGF-A水平升高,ONOO^-减少,e NOS和Akt磷酸化水平提高;高浓度硝酸甘油组EPCs活力下降,细胞ROS产生过多,上清液中VEGF-A减少,e NOS和Akt的磷酸化水平显著降低;联用β-巯基乙醇后可降低高浓度硝酸甘油组上清液中ONOO^-的含量,提高p-Akt和p-e NOS水平,增强EPCs的活力。结论:硝酸甘油可以通过PI3K/Akt/e NOS信号通路影响冠心病患者EPCs的活力,联用β-巯基乙醇可通过改善内皮祖细胞内的氧化应激并激活PI3K/Akt/e NOS信号通路来改善高浓度硝酸甘油引起的耐药。AIM： To observe the effects and mechanisms of nitroglycerin（ NTG） on cell viability and β-mercaptoethanol（ β-ME） on ameliorating nitrate tolerance of peripheral blood-derived endothelial progenitor cells（ EPCs） in coronary heart disease（ CAD） patients. METHODS： We studied 75 patients with diagnosis of coronary artery disease who were assigned to control group and NTG group. EPCs were evaluated by flow cytometry. Vascular endothelial growth factorA（ VEGF-A） and peroxynitrite anion（ ONOO^-） production were measured by ELISA. EPCs were cultured in vitro with NTG and β-ME stimulation. The cell viability was determined by MTT assay. The levels of VEGF-A,ONOO^- and reactive oxygen species（ ROS） were measured by ELISA and DCFH-DA assay. The protein levels of Akt,p-Akt,endothelial nitric oxide synthase（ e NOS） and p-e NOS were determined by Western blot. RESULTS： Compared with the control group,the circulating EPCs levels were significantly lowered,plasma ONOO^- production was vitally increased,but there was a markedly decrease of VEGF-A production in the patients treated with excess NTG（ P〈0. 05）. Moderate dose of NTG increased the viability of EPCs,VEGF-A production,and phosphorylated protein levels of Akt and e NOS. Excess NTG was shown to reverse the effect of moderate dose of NTG,but β-ME improved the adverse effect of excess NTG. CONCLUSION： Moderate dose of NTG effectively promotes EPCs viability by PI3 K/Akt/e NOS signaling pathway and β-ME improves NTG-induced tolerance by reducing oxidative stress and up-regulating the PI3 K/Akt/e NOS signaling pathway.

关 键 词：内皮祖细胞 硝酸甘油 细胞活力 血管内皮生长因子A PI3K/Akt/eNOS信号通路 β-巯基乙醇 

分 类 号：R541.4[医药卫生—心血管疾病] R363[医药卫生—内科学]