脐血内皮祖细胞的分离和培养

目的:研究从脐带血中分离内皮祖细胞的分离、培养及向内皮细胞方向诱导分化方法和条件。方法:从新鲜脐血中用密度梯度离心方法分离出单核细胞,在添加VEGF的M199培养液中培养,每隔3-4 d换一次液。培养16 d后,消化贴壁细胞,用DiI-ac-LDL及FITC-UEA-1对其进行免疫荧光及流式细胞仪分析。结果:培养3-4 d单核细胞开始贴壁,14 d左右开始出现索条状结构,免疫荧光鉴定显示贴壁细胞呈双荧光染色阳性,流式细胞仪分析显示70%贴壁细胞表达FITC-UEA-1, 85%贴壁细胞表达DiI-ac-LDL。结论:脐血中富含内皮祖细胞,在一定的培养条件下,可分化为内皮样细胞。

The Isolation and Culture of Endothelial Progenitor cells in Umbilical Blood

Objective:To investigate how to isolate and culture endothelial progenitor cells and how to make it differentiate into endothelial cells. Methods: mononuclear cells were isolated from umbilical blood with Ficoll-paque by density gradient centrifu gation. MNCs were cultured in M199 with the supplement of VEGF,b-FGF,IGF-1. Every 3-4 days culture media was changed. After 16 days of culture,attached cells were detached, stained with DiI-LDL and FITC-UEA and were be observed under flu romicroscope. Simultaneously,MNCs were analyzed with flow cytometer. Results: Attached cells were observed after 3 or 4 days of culture and cord-like structure were noticed after 16 days or so. According to immunofluorescence,more than 90% attached cells were double positive fluorescence. Analyzed by flowcytometry,70% attached cells bound FITC-UEA and more than 90% attached cells uptook Dii-LDL. Conclusion:Endothelial progenitor cells enriched in umbilical blood and can differentiate into endothelial cells under certain conditions.