过氧亚硝基阴离子对实验小鼠骨髓源内皮祖细胞的损伤作用  

Peroxynitrite induce Mice Bone Marrow Derived EPCs Injury in Vitro

作　　者：刘腾[1] 侯丹丹[1] 赵倩[1] 王可[1] 马璐[1] 李笑[1] 徐海波[1] 王雯[1] 

机构地区：[1]首都医科大学基础医学院生理与病理生理学系,北京100069

出　　处：《微循环学杂志》2012年第4期1-4,104,F0003,I0001,共7页Chinese Journal of Microcirculation

基　　金：北京市自然科学基金(7112012);心血管重塑相关疾病教育部省部共建重点实验室开放课题(2012XXGB01)

摘　　要：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。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 immunohistochemistry 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.

关 键 词：内皮祖细胞 过氧亚硝基 损伤作用 小鼠骨髓 内皮细胞功能 离子对 心血管疾病 实验 

分 类 号：R363.2[医药卫生—病理学]