人外周血来源的过度生长内皮细胞的分离、培养及鉴定

与内皮干细胞相比,外周血来源的过度生长内皮细胞（blood outgrowth endothelial cells,BOECs）富含血管新生和细胞黏附所需的蛋白,生物学特性更像内皮细胞;同时它克服了成熟的人脐血内皮细胞体外培养扩增量少和表型易发生改变的缺点,成为一种研究血管异常相关性疾病的新工具。本研究旨在建立从人外周血中体外培养、扩增BOECs,并进行鉴定的方法。采集外周血,梯度离心获得单个核细胞,接种于胶原铺板的细胞培养皿上,EGM-2培养4周。观察BOECs的形态学特征,流式细胞术分析细胞表面抗原表达。血管性血友病因子（von Willebrand factor,vWF）多聚体分析BOECs上清v WF多聚体分布,荧光共聚焦显微镜鉴定细胞内v WF的贮存及vWF的刺激分泌。结果表明,体外诱导培养4周左右,出现克隆性生长的细胞集落,BOECs呈现＂铺路石＂样外观。经过3周左右的扩增后,过度生长的内皮细胞表达CD31、CD34、EPCR阳性,CD14、CD45、CD133阴性。收集BOECs上清,进行v WF多聚体分析,与正常血浆相比v WF多聚体分布无差异。在荧光共聚焦显微镜下观察,BOECs内贮存vWF,加入佛波酯（phorbol-12-myristate-13-acetate,PMA）刺激后,细胞内v WF增加,细胞表面可见vWF丝状结构。结论：本研究运用该实验方法在国内首次实现了人BOECs的体外培养、扩增及鉴定。该方法可以为血管性血友病病人发病机制的探讨提供天然的细胞模型,并可能为病人基因治疗提供新工具。

Isolation,Culture and Characterization of Outgrowth Endothelial Cells from the Human Peripheral Blood

Compared with endothelial progenitor cells, outgrowth endothelial cells （BOECs） from peripheral blood are rich in protein for blood angiogenesis and cell adhesion, similar to mature endothelial cells in biological characteristics. Moreover, they are now replacing human umbilical vein endothelial cells for the latter＇s limited life span and drift of phenotype, and might become a new tool for exploring the vascular abnormalities. This study was aimed to establish the protocol of producing BOECs, and then analyze the cell phenotype and function of BOECs. Mononuclear cells were collected from peripheral blood by gradient centrifugation and then seeded on plates and cultivated in EGM-2 medium for 4 weeks. The morphological changes of cells were observed and cell phenotype was examined by flow cytometry. VWF mummers were used to analyse the distribution of vWF multimers in supemant of BOECs and the storage of vWF in BOECs, and the secretion of vWF in BOECs under stimulation was detected by confocal fluorescence microscopy. The results showed that after 4-week-culture in vitro, the cell colonies and characteristic cobblestone-like morphology of BOECs were found in plates. For another three weeks of expansion, BOECs expressed CD31, CD34, and EPCR, without the expression of CD14, CD45 and CD133. The vWF from BOECs cell supematant shared the same multimer pattern as that in normal plasma. By confocal fluorescence microscopy, vWFs were observed in BOECs. The amount of vWF increased in cells, and vWF strings were formed on cell surface by the stimulation of phorbol-12-myristate-13- acetate（PMA）. It is concluded that the BOECs are first succeesssfuUy established, and the phenotype and function of BOECs are analyzed. They are the native cell models for the pathogenesis of von Willebrand diseases （ vWD）, and may be used as new gene therapy tools for vWD.