中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (10): 1733-1736.doi: 10.3969/j.issn.1673-8225.2012.10.006

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

大鼠骨髓内皮祖细胞的分离培养与鉴定★

王  丽1，张会峰2，袁慧娟2，马跃华2，汪艳芳2，虎子颖2，苏  永2，赵志刚2   

  1. 1郑州大学第一附属医院内分泌科，河南省郑州市 450052；2河南省人民医院二内分泌科，河南省郑州市  450003
  • 收稿日期:2011-12-07 修回日期:2012-01-19 出版日期:2012-03-04 发布日期:2012-03-04
  • 通讯作者: 赵志刚，主任医师，教授，河南省人民医院二内分泌科，河南省郑州市 450003 Zhaozhigang1957@126.com
  • 作者简介:王丽★，女，1985年生，山东省临沂市人，汉族，郑州大学在读硕士，主要从事糖尿病并发症的防治与治疗方面的研究。 yishipiaoguo@163.com

Separation, culture and identification of rat bone marrow-derived endothelial progenitor cells

Wang Li1, Zhang Hui-feng2, Yuan Hui-juan2, Ma Yue-hua2, Wang Yan-fang2, Hu Zi-ying2, Su Yong2, Zhao Zhi-gang2   

  1. 1Department of Endocrinology, First Affiliated Hospital of Zhengzhou University, Zhengzhou  450052, Henan Province, China; 2Second Department of Endocrinology, Henan Provincial People's Hospital, Zhengzhou  450003, Henan Province, China
  • Received:2011-12-07 Revised:2012-01-19 Online:2012-03-04 Published:2012-03-04
  • Contact: author: Zhao Zhi-gang, Chief physician, Professor, Second Department of Endocrinology, Henan Provincial People's Hospital, Zhengzhou 450003, Henan Province, China Zhaozhigang1957@126.com
  • About author:Wang Li★, Studying for master’s degree, Department of Endocrinology, First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, China yishipiaoguo@163.com

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摘要：

背景：内皮祖细胞参与血管新生，但其分离、培养、鉴定方法目前并不统一。
目的：探索大鼠骨髓内皮祖细胞的分离培养及鉴定方法。
方法：使用密度梯度离心法及差速贴壁法联合的方法培养内皮祖细胞，在倒置显微镜下观察细胞生长情况及形态变化，使用Dil标记的乙酰化低密度脂蛋白和FITC标记的荆豆凝集素1双荧光染色、流式细胞仪检测细胞表面抗原CD34、CD133表达情况。
结果与结论：培养前4 d细胞增殖不明显，第5~10天迅速增殖，并可见细胞集落及线状结构形成。培养第7天的内皮祖细胞具有吞噬Dil标记的乙酰化低密度脂蛋白和FITC标记的荆豆凝集素1的功能。流式细胞仪检测体外培养第10天的细胞，CD133+ 细胞占19.2%，CD34+ 细胞占28.7%，CD34+/CD133+ 细胞占19.1%。说明密度梯度离心法联合差速贴壁法可在体外有效分离培养大鼠骨髓内皮祖细胞。
关键词：内皮祖细胞；细胞分离；细胞培养；骨髓；细胞鉴定
doi:10.3969/j.issn.1673-8225.2012.10.006

关键词: 内皮祖细胞, 细胞分离, 细胞培养, 骨髓, 细胞鉴定

Abstract:

BACKGROUND: Endothelial progenitor cells (EPCs) are involved in angiogenesis, but its isolation, culture and identification methods are not uniform currently.
OBJECTIVE: To explore the methods of separation, culture and identification of rat bone marrow-derived EPCs.
METHODS: EPCs were cultured using density gradient centrifugation and differential velocity adherent methods, growth and morphology changes of cells were observed under inverted microscope. The expression of cell surface antigen CD34 and CD133 were detected by using Dil labeled acetylated low-density lipoprotein (Dil-ac-LDL) and FITC-labeled Ulex europaeus agglutinin 1 (FITC-UEA-1) double dyeing and flow cytometry.  RESULTS AND CONCLUSION: For the first four days, cell proliferation was not obvious, at the 5-10 days, cell proliferation was obvious, and colony-forming unit (CFU) and lineage structure could be seen. At the 7th day, EPCs could swallow the function of DiL-ac-LDL and FITC-UEA-1. At the 10th day, in vitro flow cytometry detection showed that the CD133+ cells were accounted for 19.2%, CD34+ cells were accounted for 28.7%, CD34+/CD133+cells were accounted for 19.1%. Using density gradient centrifugation and differential velocity adherent methods can separate and culture bone marrow derived EPCs.

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