人外周血内皮祖细胞培养方法的建立

背景：成人外周血来源丰富，但内皮祖细胞含量较少，为使其能够更好的应用于组织工程及细胞治疗，有必要建立外周血内皮祖细胞成熟、稳定的体外扩增体系。目的：建立稳定的人外周血分离、培养和体外扩增血管内皮祖细胞的方法。方法：应用密度梯度离心法，获取外周血单个核细胞，将分选后细胞接种于预先包埋了人纤维连接蛋白的培养板上，加入内皮祖细胞专用培养基中培养3 d后，洗掉非贴壁细胞，培养至第6天，收集贴壁细胞，应用倒置显微镜和苏木精-伊红染色进行细胞形态学观察；采用MTT法和细胞计数测定第1，3代细胞生长曲线；应用流式细胞仪测定祖细胞和内皮细胞系标志，对培养的细胞进行鉴定。结果与结论：细胞生长曲线测定表明接种后第3天细胞进入指数增生期，至第6天进入平台期，随着传代次数的增加，细胞增殖速度变慢，同时表达干细胞表面标志CD34、CD133和内皮细胞表面标志血管性血友病因子、血管内皮生长因子受体2。证明人外周血可以分离培养内皮祖细胞。

Cultivation of endothelial progenitor cells from human peripheral blood

BACKGROUND:Although peripheral blood in adults can provide abundant source of cells, the content of endothelial progenitor cells is low. It is necessary to establish a stable and mature in vitro cultivation system of endothelial progenitor cells in order to achieve a better application in tissue engineering and cell therapy.OBJECTIVE:To establish a stable cultivation system of endothelial progenitor cells from human peripheral blood in vitro, including cell isolation, culture and expansion.METHODS:Mononuclear cells were isolated from the human peripheral anticoagulant blood sample by density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. The cells were cultured in endothelial progenitor medium. Non-adherent cells were washed away. The adherent cells were collected at the 6th day after cultivation. The morphologic changes of endothelial progenitor cells were observed by hematoxylin-eosin staining through invert microscope. Growth curves of cells of passages 1 and 3 were measured by MTT and cell counting. The cultured cells were identified by flow cytometry based on the presence of specific surface markers in progenitor cells and endothelial cell line.RESULTS AND CONCLUSION:According to the growth curves, cells entered logarthmic growth phase at the 3rd day after inoculation, and then entered platform phase at the 6th day after inoculation. Cell proliferation rate slowed with increasing passage times. The stem cell surface markers CD34, CD133 and endothelial cell surface markers von willebrand factor and vascular endothelial growth factor receptor 2 were expressed at the same time. These results demonstrate that endothelial progenitor cells derived from human peripheral blood can be propagated in vitro.