巴曲酶对内皮祖细胞功能的影响及其机制的探讨

Effect of batroxobin on the activity of endothelial progenitor cells and its possible mechanism in vitro

目的研究巴曲酶(DF-521)对体外培养下内皮祖细胞(EPCs)的增殖、分化、成血管能力以及NO分泌能力的影响,并探讨其可能的机制。方法从人外周血中提取单个核细胞,用流式细胞术检测细胞表型CD34、CD31、血管内皮生长因子2和血管性假血友病因子(vWF),激光共聚焦显微镜观察细胞经过FITC标记的荆豆凝集素I和DiI标记的乙酰化低密度脂蛋白双染色后的情况,由此鉴定EPCs。实验分为:DF-521的低、中、高剂量干预组和对照组。检测各组EPCs的CD31和vWF表达,观察EPCs在基质胶上的成管情况以及测定培养液中NO的浓度。透射电子显微镜鉴定分化后的细胞。结果低、中、高剂量干预组EPCs的数量、CD31、vWF表达及NO浓度均明显高于对照组(P<0.05),而且这些效应随DF-521浓度升高而增加(P<0.05)。高剂量干预组EPCs形成条梭状和管腔样结构。电子显微镜观察到怀布尔-帕拉德小体。结论体外培养务件下.DF-521促进EPCs的增殖和成血管功能,并诱导其向内皮细胞分化,改善分泌NO的能力。DF-521的这些作用可能与内皮型一氧化氮合酶有关。

Objective To investigate the effect of batroxobin（DF-521）on the number and the activity of endothelial progenitor ceils （EPCs） in vitro and its possible mechanism. Methods The mononuclear cells were collected from the human peripheral blood,isolated by the density gradient centrifugation and cultured. On the 6th day, the phenotypes（CD34, CD31, VEGF-2, vWF） of the attached cells were assayed by flow cytometry（FCM）. The cells were observed by the laser scanning confocal microscope after double staining with FITC-UEA-Ⅰ and DiI-acLDL. After identified,the EPCs were cultured in three kinds of nutrient liquid which included different doses of DF-521. The phenotypes（CD31 ,vWF） of EPCs were assessed on the 14th and 21st day respectively by FCM. The proliferation and angiogenesis of EPCs in vitro were analyzed by cell counting and tube formation. NO concentrations were measured with the nitrate reductase method. The ultrastructure of the cells was observed with a transmission electron microscope（TEM）. Results The number,expression of CD31 and vWF and the NO concentration in EPCs were remarkably increased after treatment with DF-521 compared with those in the control （P 〈 0.05）. Furthermore,these effects had dose-dependent manner （P 〈 0.05）. In the high dose group, the EPCs formed shuttle-like and tube-like structures after being plated in matrigel. The W-P bodies could be found in the cells by the TEM. Conclusion The proliferation,angiogenesis,differentiation into endothelial cells and the activity of excreting NO of EPCs can be improved by DF-521 in vitro . The endothelial nitric oxide synthase may play a key role in these effects.