兔全骨髓培养诱导分化获取内皮祖细胞
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国家自然科学基金青年科学基金项目


Expansion of endothelial progenitor cells from rabbit bone marrow culture
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    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扩增能力。方法 新西兰兔10只,每只兔骨髓穿刺抽取骨髓3ml,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,绘制细胞生长曲线评估扩增能力。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。

    Abstract:

    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.

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  • 收稿日期:2010-12-29
  • 最后修改日期:2011-02-27
  • 录用日期:2011-03-01
  • 在线发布日期: 2011-03-17
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