| 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。 |
| 关键词: 骨髓 内皮细胞 干细胞 兔 细胞分选 |
| DOI：10.3724/SP.J.1008.2011.0243 |
| 投稿时间：2010-12-29修订日期：2011-02-27 |
| 基金项目:国家自然科学基金青年科学基金项目 |
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| Expansion of endothelial progenitor cells from rabbit bone marrow culture |
| FANG Xing-gen1,2, ZHAO Rui1, LI Zi-fu1, YANG Peng-fei1, HUANG Qing-hai1, XU Yi1,HONG Bo1, LIU Jian-min1* |
| (1. Department of Neurosurgery, Changhai Hospital, Second Military Medical University, Shanghai 200433, China; 2. Department of Neurosurgery, Yijishan Hospital, Wannan Medical College, Wuhu 241000, Anhui, China) |
| 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. |
| Key words: bone marrow endothelial progenitor cells stem cells rabbit Cell Separation |
