小鼠骨髓来源内皮祖细胞的分离、培养及鉴定

Isolation,culture and characterization of bone marrow derived endothelial progenitor cells from mice

目的:探讨自小鼠骨髓分离培养内皮祖细胞(endothelial progenitor cells,EPC)的方法及其鉴定和生物学特性。方法:密度梯度离心法分离获取小鼠骨髓单核细胞,培养于EGM-2 SingleQuots培养液中,每天于倒置显微镜下观察EPC的形态。应用细胞免疫荧光法、流式细胞技术鉴定EPC表面抗原标志CD34、血管内皮生长因子受体-2(VEGFR-2),荧光显微镜检测EPC吞噬DiI标记的乙酰化低密度脂蛋白(DiI-AC-LDL)及结合FITC标记的荆豆凝集素(FITC-UEA-lectin)功能。结果:早期EPC呈长梭形,细胞群落可呈现线状、管状、网状生长状态,培养7 d后出现干细胞集落,并逐渐增多;培养3周后,细胞集落逐渐消失,细胞呈现铺路石样生长状态。培养获得的EPC绝大部分可同时表达细胞表面标记物CD34及VEGFR2,并具有吞噬DiI-AC-LDL及结合FITC-UEA-lectin功能。结论:自小鼠骨髓中分离、培养、纯化EPC是一种方便、经济、高效的培养方法。通过该方法培养获得的EPC增殖能力强,数量充足,生物学特性稳定。该方法对今后利用EPC进行细胞移植治疗的相关实验有着重要意义。

Objective： To investigate the isolation and culture methods of endothelial progenitor cell（EPC） from mice bone marrow, explore its identification methods and biological characteristics. Methods： Mice bone marrow was harvested and centrifuged for bone marrow mononuclear cells （BMMNCs） which were later seeded in the cell culture flasks and cultured with EGM-2 SingleQuots. Cultured cells were observed by inverted microscope every day. Cultured EPCs were verified by cell surface antigens CD34 and VEGFR2 through immunofluorescence and flow cytometer. The abilities of uptake DiI- Ac- LDL and binding FITC- UEA- 1 of cultured cells were also tested. appearance. Cell colonies began to appeared since the 7th day of culture and increased gradually. Since about the 3nd weeks of culture, the cells declined gradually and changed to be a cobblestone-like appearance. Cultured EPCs co-expressed surface antigens CD34 and VEGFR2 and were able to uptake DiI-Ac-LDL and bind FITC-UEA- lectin. Conclusion： Our method of culturing EPC is feasible, economical and high-efficient. The EPCs we cultured have a high potential of proliferation, abundant cell count and stable biological functions. Our method of culturing EPC should be important for the later EPC-based cell transplantation experiments.