兔血管内皮祖细胞的分离及诱导分化

Isolation and inducement of endothelial progenitor cells from rabbit bone

目的：探讨自兔骨髓中分离及诱导血管内皮祖细胞（EPCs）的方法。方法：抽取兔骨髓，Ficoll离心液梯度离心，分离出骨髓单个核细胞，用含有血管内皮生长因子（VEGF）、碱性纤维细胞生长因子（bFGF）、胰岛素样生长因子-1（IGF-1）的M199培养液诱导培养EPCs，透射电镜及免疫荧光法对所培养细胞进行鉴定。结果：细胞诱导培养48-72h后即可见有细胞贴壁，逐渐变成梭形。培养至8d左右，可观察到数条由梭形贴壁细胞直线生长连接而成的条索，7-10d出现多个细胞团，贴壁的梭形细胞开始从细胞团的边缘出芽长出，培养至14d左右，细胞逐渐融合，连接成大片条索状结构。透射电镜及免疫荧光检查证实为血管内皮祖细胞。结论：梯度离心法白兔骨髓获得单个核细胞，在体外经诱导分化可获得血管内皮祖细胞。

Objective：To investigate the method of isolation and inducement of endothelial progenitor cells from rabbit bone marrow. Method： Rabbit bone marrow was obtained by iliac crest puncture, then bone marrow mononuclearcells were isolated by gradient centrifugation using Ficoll isolative liquid. Mononuclearcells were then cultivated with M199 culture medium and induced to EPCs by VEGF, bFGF and IGF-1. Cultivated cells were identified by immunofluorescence and electron microscope. Result： Attached cells appeared after 48-72 hours culture and become fusiform shape gradually. Attached cells formed several cord-like structure after 8 days culture, which is the character of endothelial cells. Cell clusters were observed after 7-10 days from which attached cells were budding. Cells mixed together after 14 days and were proved to be EPCs by electron microscope and Immunohistochemistry results. Conclusion：The mononuclear cells isolated form rabbit bone marrow by gradient centrifugation can be induced into endothelial progenitor cells in vitro.