血管紧张素Ⅱ对外周血内皮祖细胞的影响

Effects of angiotensinⅡ on peripheral blood endothelial progenitor cells

目的 观察血管紧张素Ⅱ (AngⅡ )对外周血内皮祖细胞 (EPCs)数量和功能的影响。方法 密度梯度离心法获取外周血单个核细胞 (MNCs) ,培养 7d后 ,收集贴壁细胞并加入不同浓度AngⅡ (10 -3 mol/L、10 -5mol/L、10 -7mol/L、10 -9mol/L)干预一定时间 (6、12、2 4h和 4 8h)。多波长激光共聚焦显微镜鉴定FITC标记荆豆凝集素Ⅰ和DiI标记的乙酰化低密度脂蛋白双染色细胞为正在分化的EPCs ,流式细胞仪检测其表面标志进一步鉴定EPCs,倒置荧光显微镜下计数。分别观察EPCs的增殖、迁移、黏附和体外血管生成能力 ,并观察缬沙坦的影响。结果 AngⅡ促进外周血EPCs扩增 ,10 3 mol/LAngⅡ作用 2 4h对EPCs数量的影响最为显著 (P <0 0 1)。AngⅡ也显著增强了外周血EPCs的黏附、迁移、增殖和体外血管生成能力。而缬沙坦可显著抑制AngⅡ的这些作用。结论 AngⅡ可通过血管紧张素 1受体介导增加EPCs数量、改善其功能 ,并呈浓度和时间依赖性。

Objective To investigate the effect of AngiotensinⅡ(AngⅡ) on the quantity proliferation, migration and adhesion of endothelial progenitor cells (EPCs).Methods Total mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation and then the cells were plated on fibronectin coated culture dishes. After 7 days of culture, several groups of attached cells were incubated with AngⅡ(to make a series of concentrations: 10 -3 mol/L,10 -5 mol/L,10 -7 mol/L,10 -9 mol/L) or vehicle control for the respective time points (6 , 12 , 24 h and 48 h). In other groups of EPCs,valsartan(1×10 -5 mol/L)and AngⅡ were added to the culture medium together.EPCs were characterized as adherent cells double positive for DiLDL uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. EPCs were further documented by demonstrating the expression of KDR, VEGFR 2 and AC133 with flow cytometry. EPCs proliferation, migration and in vitro vasculogenesis activity were assayed with MTT assay, modified Boyden chamber assay and in vitro vasculogenesis kit, respectively. EPCs adhesion assay was performed by replating MNCs on fibronectin coated dishes.Results Incubation of isolated human MNCs with AngⅡ increased the number of EPCs, with a maximum at 10 -3 mol/L after 24 hours ( P <0 01). In addition, AngⅡ promotes EPCs proliferative, migratory, adhesive and in vitro vasculogenetic capacity. The effect of AngⅡwas blocked by pretreatment of valsartan.Conclusions It is suggested that angiotensinⅡ may promote EPCs augmentation and enhance its functional activity through angiotensin receptor.