Objective: To investigate the effect of peroxynitrite (ONOO-) on mouse bone marrow derived endothelial progenitor cells (EPCs). Method: EPCs were isolated from mouse bone marrow via ficoll density gradient centrifugation, and seeded in EGM-2 medium. EPCs were identified by expression of VE-cadherin, VEGFR-2 and CD133, incorporation of Dil-ac-LDL and binding of FITC-UEA-1 with immunofluorescence staining. Cells from passages 2 to 3 were used in this study. After treated with different dose of ONOO" donor 3-morpholinosydnonimine (SIN-1), EPCs viability was detected by MTT assay to determine an effective challenging dose. After treated with SIN-1 and ONOO" scavengers FeTMPyP, EPCs viability and apoptosis were measured by MTT and TUNEL assay respectively. Besides, the expression of 3-nitrotyrosine (NT) , imprinting of endogenous nitrative stress, was determined by immunohisto-chemistry staining. Results; 800μM SIN-1 significantly decreased cell viability and induced apoptosis of EPCs, while 10μM ONOO- scavengers FeTMPyP could reverse these changes. There was an obvious increase of NT expression in EPCs after SIN-1 stimulation, which was also inhibited when pretreated with FeTMPyP. Conclusion: The ONOO- produced by SIN-1 can induce EPCs apoptosis through enhancing nitrative stress in vitro, FeTMPyP can reverse these changes.%目的:观察过氧亚硝基阴离子(ONOO-)对实验小鼠骨髓源内皮祖细胞(EPCs)的体外损伤作用.方法:无菌抽取4周龄雄性C57小鼠骨髓,密度梯度离心法分离获取单个核细胞,EGM-2培养基诱导培养EPCs.用免疫荧光染色法进行EPCs表面标志物检测和功能试验鉴定EPCs,培养2~3代后用于实验.通过不同浓度的3-吗啉斯德酮亚胺(SIN-1,CONOO-供体)作用于EPCs 24h,确定SIN-1的有效作用剂量.将EPCs分为空白对照组、SIN-1组和FeTMPyP(ONOO-清除剂)干预组,分别用MTT法检测各组EPCs存活率,TUNEL法检测EPCs凋亡,免疫荧光染色法检测细胞内ONOO-生成印记——3-硝基酪氨酸(NT)表达情况.结果:上述培养细胞表面标记物和功能试验均阳性,是EPCs.800μM SIN-1刺激24h后EPCs存活率较空白对照组明显下降(P<0.05),10μM FeTMPyP干预能抑制这种作用.SIN-1刺激后EPCs凋亡率较空白对照组明显增加(P<0.05),FeTMPyP干预能抑制SIN-1诱导的EPCs凋亡.SIN-1刺激EPCs后细胞内NT表达较空白对照组明显增加(P<0.05),FeTMPyP干预能减少NT的表达.结论:SIN-1能够介导硝化应激,诱导EPCs凋亡,造成EPCs损伤,FeTMPyP干预能抑制SIN-1的上述作用.
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