兔全骨髓培养诱导分化获取内皮祖细胞

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。

Expansion of endothelial progenitor cells from rabbit bone marrow culture

Objective Rabbit endothelial progenitor cells were harvested from culturing bone marrow with EGM-2MV (n=10), and efficacy of the cells expansion was observed. Methods Three millimeter bone marrow of each rabbit was cultured with EGM-2MV medium in flasks coated with fibronectin. Morphology was observed with phase contrast microscopy, and a growth curve was constructed to evaluate the efficacy of expansion. Twelve days later, the attached cells were identified with immunocytochemical staining for CD133, CD34, vascular endothelial growth factor receptor 2(VEGFR-2). The function of cells was tested by evaluating the ability of incorporation of Dil-ac-LDL and combining FITC-UEA-1 lectin. Meanwhile the attached cells were selected with CD133 immunomagnetic beads. The percentage of CD133, CD34, and VEGFR-2, CD34/ VEGFR-2 and CD133/ CD34/ VEGFR-2 antigen-positive cells were calculated and compared using flow cytometry before and after immunomagnetic beads selection. Results The cluster-like or colony-like growth cells were found 48 h after bone marrow cultured. The cells were spindle, triangle or polygon shaped. The cells growth curve showed S type. About (1.51±0.29) ×106 attached cells were harvested from each sample twelve days later. The cells took up Dil-ac-LDL and combined with FITC-UEA-1 lectin. Immunocytochemistry showed that CD34, CD133, and VEGFR-2 were positive. The percentage of CD34+/ VEGFR-2+ and CD133+/CD34+/ VEGFR-2+ cells after immunomagnetic beads selection were 3.38 and 6.14 times higher than those of unselected cells, respectively. Conclusion Endothelial progenitor cells can be harvested directly from rabbit bone marrow in a simple and effective method.