摘要
目的研究大鼠骨髓来源的内皮祖细胞（EPC）的分离、鉴定、培养方法以及向内皮细胞分化的诱导条件。方法密度梯度离心法分离SD大鼠股骨及胫骨单个核细胞，经差速贴壁后取2次贴壁细胞置于纤连蛋白铺被的培养板中。在血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）及表皮生长因子（EGF）诱导下培养两周，观察其形态学改变，以免疫细胞化学染色及Dil-acLDL、FITC-UEA-1双荧光染色法对其进行鉴定。结果贴壁细胞呈现铺路石、团簇样生长，呈梭形、线样排列特殊形态。免疫细胞化学结果显示，贴壁细胞CD133、CD34、Flk-1、血管性假血友病因子（vWF）在不同时段呈阳性表达。诱导培养第2天，CD133阳性表达率为（79．4±4．5）％。自诱导培养的第6天开始，Flk-1与vWF呈高表达，阳性率为（74．2±3．5）％和（72．2±4．3）％。结论用Percoll密度梯度离心法在VEGF、bFGF及EGF培养体系下可以获得较高纯度的EPC，该细胞具有内皮细胞的特性，经过体外诱导可以分化为内皮细胞。
Objective To investigate the methods for isolating, identifying and culturing endothelial progenitor cells （EPCs） from rat bone marrow, as well as the differentiation of EPCs into endothelial cells. Methods The mononuclear cells were isolated from rat femoral and tibial bone marrow using percoll density gradient centrifugation, then plated on dishes coated with fibronectin, and induced with vascular endothlial growth factor （VEGF）, basic fibroblast growth factor（bFGF） and epidermal growth factor （EGF） for two weeks. The expression of cell markers was assessed by immunocytochemistry, and the attached cells were stained with Dil-ac-LDL and FITC-UEA-1. Results The attached EPCs were able to line up in the typical cord-like structure and formed clusters and cobblestone. Adherent cells showed CD34, Flk- 1, CD133, vWF positive in different time and took up Dil-acLDL, bound FITC-UEA-1 double positive fluorescence by laser confocal microscopy. On day 2 of induced differentiation, CD133 positive rate was （79.4±4.5）%. Flk and vWF showed high level of expression in （74.2±3.5）% and （72.2±4.3）% of the cells respectively from day 6. Conclusions The mononuclear cells can be isolated from rat bone marrow by percoll density gradient centrifugation. After induction of VEGF, bFGF and EGF, highly purified EPCs are obtained with the same characteristics of endothelial cells. EPCs can be differentiated into endothelial cells in vitro.
出处
《中华生物医学工程杂志》
CAS
2009年第6期463-466,共4页
Chinese Journal of Biomedical Engineering
基金
国家自然科学基金面上项目（30700166、30800271）
关键词
骨髓细胞
内皮细胞
干细胞
分化
诱导
Bone marrow cells
Endothelial calls
Stem cells
Differentiation
Induction
