中文摘要：

目的探讨采用将兔全骨髓直接体外培养诱导分化的方法获取内皮祖细胞（EPCs）,同时观察EPCs的扩增能力。方法新西兰兔10只,对每只兔穿刺并抽取骨髓3 ml,用EGM-2培养基对全骨髓进行培养,观察细胞生长及形态变化,绘制细胞生长曲线并评估其扩增能力。对培养12 d后的贴壁细胞行CD133、CD34、VEGFR-2三抗原免疫组化鉴定及吞噬乙酰化低密度脂蛋白（ac-LDL）和结合荆豆凝集素（UEA-1 lectin）的内皮细胞功能鉴定;同时对其行CD133免疫磁珠分选及流式细胞术检测,比较分选前后的CD34＋/VEGFR-2＋和CD133＋/CD34＋/VEGFR-2＋细胞比例的差异。结果全骨髓直接培养48 h后可见细胞呈丛状或集落样生长,细胞呈梭形、三角形、多边形,细胞生长曲线呈＂S＂型。经过12 d的培养,每3 ml骨髓可以获得（1.51±0.29）×106个贴壁生长的EPCs,细胞呈铺路石样外观;检测发现CD133、CD34、VEGFR-2三抗原阳性表达,并具有吞噬ac-LDL和结合荆豆凝集素（UEA-1 lectin）的内皮细胞功能。经CD133免疫磁珠分选后CD34＋/VEGFR-2＋和CD133＋/CD34＋/VEGFR-2＋的细胞比例数分别为分选前的3.38倍和6.14倍。结论通过全骨髓直接培养诱导分化获取兔EPCs的方法简单、可行。

英文摘要：

Objective To obtain endothelial progenitor cells（EPCs） by culturing total bone marrow of rabbits with EGM-2MV,and to observe the proliferation of EPCs.Methods Three millimeter bone marrow was obtained from each of the 10 New Zealand rabbits and was cultured with EGM-2MV medium in flasks coated with fibronectin.Morphology and growth of cells were observed under phase contrast microscope,and a growth curve was plotted to evaluate cell proliferation ability.Twelve days after culture,the attached cells were identified by immunocytochemical staining for CD133,CD34,and vascular endothelial growth factor receptor 2（VEGFR-2）.The functions of cells were tested by evaluating the incorporation ability of Dil-ac-LDL and the binding ability with FITC-UEA-1 lectin.The attached cells were also selected by CD133 immunomagnetic beads and flow cytometry.The percentages of CD133,CD34,and VEGFR-2,CD34/VEGFR-2 and CD133/CD34/VEGFR-2 antigen-positive cells were calculated and compared before and after immunomagnetic beads selection.Results Cluster-like or colony-like cells were grown from the bone marrow culture 48 h later;the cells were spindle,triangle or polygon in shape.The cells growth curve was ＂S＂ shaped.About（1.51±0.29）×106 attached EPCs were harvested from each sample after 12 days’ of culture;the cells took on a cobblestone appearance;they also incorporated Dil-ac-LDL and combined with FITC-UEA-1 lectin.Immunocytochemistry showed the cells were positive for CD34,CD133,and VEGFR-2.After immunomagnetic beads selection the percentages of CD34＋/VEGFR-2＋ and CD133＋/CD34＋/VEGFR-2＋ cells were 3.38 and 6.14 times higher than those before selection,respectively.Conclusion Direct culture of rabbit bone marrow is a simple and effective method to obtain EPCs.