人外周血内皮祖细胞的分离、培养及鉴定

背景：内皮祖细胞是成熟内皮细胞的前体细胞，具有新生血管和新生内皮化作用，在许多方面均有广泛应用前景，但其生物学特征及鉴定方法仍存争议。目的：探索从人外周血分离培养内皮祖细胞的方法并鉴定其生物学特征。方法：外周采血后应用密度梯度离心法分离成人外周血单核细胞，在内皮细胞全培养基重悬后接种于纤维连接蛋白包被的培养瓶中，体外培养扩增获取人内皮祖细胞并观察其形态变化、生长增殖潜能及细胞表面抗原表达情况，并通过细胞一氧化氮分泌功能测定及体外血管形成实验检测其功能学特征。结果与结论：体外诱导培养后，六七天形成纺锤样细胞簇，两三周黏附细胞发育形成鹅卵石样外观细胞，逐渐融合呈外生性生长。在相同培养条件下，与人主动脉内皮细胞相比人内皮祖细胞具有高的增殖潜能。人内皮祖细胞表达CD31、CD34、CD144、KDR，表现为典型内皮细胞系表型，此外细胞可摄取ac-LDL并结合UEA-I。在功能上内皮祖细胞可分泌一氧化氮并可在Matrigel中形成管腔样结构。提示通过密度梯度离心法分离人外周血单核细胞体外黏附诱导培养可获取人外周血内皮祖细胞；细胞形态、增殖能力、生物表型特征结合细胞功能学的综合性鉴定方法用于内皮祖细胞的鉴定具有一定意义。

Isolation, culture and characterization of endothelial progenitor cells from the human peripheral blood

BACKGROUND： Endothelial progenitor cells, known as the precursor cells of mature endothelial cells, have the function of neovascularization and neoendothelialization. Therefore, endothelial progenitor cells have potential applicability in many fields. Endothelial progenitor cells can be isolated and cultured from different resources with different methods, but the biological properties and identification of endothelial progenitor cells still have controversies. OBJECTIVE： To explore the methods of isolation and culture of endothelial progenitor cells from the human peripheral blood and to identify the biological features of endothelial progenitor cells. METHODS： Mononuclear cells were isolated from the human peripheral blood using density gradient centr~fugation, and the cells were resuspended in endothelial basal medium-2 supplemented with the EGM-2-MV-SingleQuots. Then, the ceils were inoculated in human fibronectin-coated culture flasks and cultured in EBM-2MV medium. The morphology of endothelial progenitor cells was observed. The proliferation potentialand surface markers of endothelial progenitor cells were characterized carefully. Furthermore, the functional properties such as nitric oxide release and tube formation on Matrigel were also evaluated. RESULTS AND CONCLUSION： While adherent cells maintained, spindle-shaped cells formed a cell cluster after 6-7 days. Then, adherent cells developed to endothelial progenitor cells with a cobblestone appearance after 2-3 weeks. The endothelial progenitor cells were confluent with an outgrowth appearance. Endothelial progenitor cells had a higher proliferation potential compared with human aortic endothelial cells under the same culture condition. Endothelial progenitor cells expressed CD31, CD34, CD144 and KDR, displaying an obvious endothelial phenotype. Endothelial progenitor cells were also found to uptake DiL-acLDL and exhibit lectin binding capability. Furthermore, endothelial progenitor cells were able to form capillary tubes on Matrigel and had the ability to release nitric oxide. Therefore, endothelial progenitor cells can be obtained from the human peripheral blood by density gradient centrifugation and adherent＇culture. A combining method for the identification of endothelial progenitor cells should be recommended.