中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (36): 6762-6766.doi: 10.3969/j.issn.2095-4344.2012.36.020

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

全骨髓培养法扩增小鼠内皮祖细胞

刘俊峰1，杜忠东1，陈 植1，关云谦2，李慎涛3   

  1. 1首都医科大学附属北京儿童医院心脏中心，北京市 100045；
    2首都医科大学附属宣武医院细胞生物学实验室，北京市 100053；
    3首都医科大学细胞生物学实验室基础医学院，北京市100069
  • 收稿日期:2011-11-28 修回日期:2011-12-15 出版日期:2012-09-02 发布日期:2012-09-02
  • 通讯作者: 杜忠东，教授，主任医师，博士生导师，首都医科大学附属北京儿童医院心脏中心，北京市 100045 duzhongdong@vip.Sohu.com
  • 作者简介:刘俊峰☆，男，1975年出生，四川省武胜县人，汉族，首都医科大学在读博士，主治医师，主要从事小儿心血管疾病研究。 liuboxuan@126.com

Whole bone marrow cells culture for expanding mouse endothelial progenitor cells

Liu Jun-feng1, Du Zhong-dong1, Chen Zhi1, Guan Yun-qian2, Li Shen-tao3   

  1. 1Department of Cardiology, Beijing Children’s Hospital of Capital Medical University, Beijing 100045, China;
    2Cell Biology Laboratory Affiliated Xuanwu Hospital of Capital Medical University, Beijing 100053, China;
    3School of Basic Medical, Capital Medical University, Beijing 100069, China
  • Received:2011-11-28 Revised:2011-12-15 Online:2012-09-02 Published:2012-09-02
  • Contact: Du Zhong-dong, Professor, Chief physician, Doctoral supervisor, Department of Cardiology, Beijing Children’s Hospital of Capital Medical University, Beijing 100045, China duzhongdong@vip.Sohu.com
  • About author:Liu Jun-feng☆, Studying for doctorate, Attending physician, Department of Cardiology, Beijing Children’s Hospital of Capital Medical University, Beijing 100045, China liuboxuan@126.com

PDF

650

摘要：

背景：对于小型实验动物，通过分离骨髓单个核细胞进行内皮祖细胞培养、扩增的方法较为繁琐。
目的：探讨采用全骨髓培养方式扩增小型动物内皮祖细胞的可行性。
方法：采用全骨髓培养分离C57BL/6小鼠内皮祖细胞，培养第7天行DiL-acLDL和FITC-UEA-1双荧光染色检测，并利用流式细胞仪检测其CD34、FLK-1表达情况。同时检测其体外血管生成能力，黏附、增殖和迁移能力。设立骨髓单个核细胞培养法为对照。
结果与结论：全骨髓培养至第2天便可见早期内皮祖细胞集落形成，第7天时可见大量短梭状内皮祖细胞，其具有吞噬DiL-acLDL及结合FITC-UEA-1的能力，内皮祖细胞在基质胶上同样能够形成血管样结构，培养至第2周晚期内皮祖细胞集落出现，迅速生长形成典型的铺路石样，并能够在体外传代培养，细胞数量、细胞表面CD34、FLK-1表达、体外黏附、增殖和迁移能力及晚期集落出现时间与对照组差异无显著性意义(P > 0.05)。说明采用全骨髓培养的方式能够实现小型动物内皮祖细胞的筛选扩增，且操作简便。

关键词: 骨髓, 内皮祖细胞, 小鼠, 扩增, 单个核细胞

Abstract:

BACKGROUND: The protocols of culturing and expanding endothelial progenitor cells (EPCs) through isolating bone marrow mononuclear cells are complex in small laboratory animals.
OBJECTIVE: To explore the feasibility of whole bone marrow cells culture for expanding EPCs.
METHODS: EPCs were separated from C57BL/6 mice by whole bone marrow cells culture, and the EPCs were detected with DiL-acLDL and FITC-UEA-1 double fluorescent staining after cultured for 7 days, and the expression of CD34 and FLK-1 was detected by flow cytometry. Furthermore, the functions of EPCs in vitro, including visualization, proliferation, adhesion and migration were assessed. The bone marrow mononuclear cells culture was used as control.
RESULTS AND CONCLUSION: The colony of early EPCs could be seen after cultured for 2 days with whole bone marrow cells culture method in vitro. On day 7, a large number of short spindle-like cells appeared, and exhibited the ability of binding with FITC-UEA-1 and phagocytosis of Dil-acLDL. The EPCs could form vessel like structure when cultured on matrigel. After cultured for about 2 weeks, the late EPCs colonies appeared and proliferated rapidly, in typical cobblestone morphology and could be passaged and cultured in vitro. There was no significant difference between two groups in the number of cells, the expression of CD34 and FLK-1 on the cell surface, in vitro adhesion, proliferation, migration abilities and the time for the colony appearance (P > 0.05). The method of whole bone marrow cells culture in vitro could select and expand EPCs effectively. The procedure is more simple, and especially suitable for studying EPCs functions in the small laboratory animals.

中图分类号: