中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (14): 2508-2512.doi: 10.3969/j.issn.1673-8225.2010.14.008

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

全骨髓贴壁并差速传代分离纯化大鼠骨髓间充质干细胞：与密度梯度离心法的比较

林楚伟，周胜华，杜优优   

  1. 中南大学湘雅二医院心内科，湖南省长沙市  410000
  • 出版日期:2010-04-02 发布日期:2010-04-02
  • 作者简介:林楚伟☆，男，1982年生，湖南省浏阳市人，汉族，中南大学在读博士，主要从事心肌梗死干细胞治疗的研究。 linchuwei@yahoo.com.cn
  • 基金资助:

    国家自然科学基金资助项目(30871053)。

Rat bone marrow mesenchymal stem cells isolated and purified by whole bone marrow adherent method and differential passage: A comparison with density gradient centrifugation

Lin Chu-wei, Zhou Sheng-hua, Du You-you   

  1. Department of Cardiology, Second Xiangya Hospital, Central South University, Changsha   410000, Hunan Province, China
  • Online:2010-04-02 Published:2010-04-02
  • About author:Lin Chu-wei☆ Studying for doctorate, Department of Cardiology, Second Xiangya Hospital, Central South University, Changsha 410000, Hunan Province, China linchuwei@yahoo.com.cn
  • Supported by:

    the National Natural Science Foundation of China, No. 30871053*

PDF

615

摘要：

背景：目前国内外分离纯化骨髓间充质干细胞有两种主要方法：密度梯度离心法及全骨髓贴壁法，前者步骤较复杂，后者简单易操作，但纯化效果不理想。
目的：在全骨髓贴壁分离骨髓间充质干细胞基础上，并用差速传代消化法，建立大鼠骨髓间充质干细胞体外分离培养纯化方法。
方法：全骨髓贴壁培养法分离并差速消化传代大鼠骨髓间充质干细胞，利用间充质干细胞在消化传代过程中较其他骨髓细胞消化悬浮速度快，以及贴壁快的特点，代替密度梯度离心操作来分离纯化间充质干细胞，对其形态学特征进行观察，并与密度梯度离心法比较两种分离培养法的细胞生长增殖情况；观察碱性磷酸酶及油红染色情况，验证骨髓间充质干细胞的分化能力；检测细胞表面标记物，验证免疫特性及检测其纯度。
结果与结论：全骨髓贴壁培养法分离并差速传代大鼠骨髓间充质干细胞，流式细胞鉴定、成骨成脂肪培养结果显示其细胞免疫特性、纯度、分化能力与密度梯度离心法无显著差异，但细胞活力，增殖能力有明显提高。

关键词: 细胞培养, 细胞分离, 间充质干细胞, 纯化, 密度梯度离心, 全骨髓贴壁, 差速传代

Abstract:

BACKGROUND: At present, there are two main methods of isolating and purifying bone marrow mesenchymal stem cells (BMSCs): density gradient centrifugation and the whole bone marrow adherent method. The former has complicated procedures, and the latter has simple operation, but the purified outcomes are not ideal.
OBJECTIVE: To establish a rat BMSCs isolated and cultured in vitro purification methods on the basis of the whole bone marrow adherence and isolation of BMSCs in combination of differential passage digestion method.
METHODS: By whole bone marrow adherent culturing to isolate and differential digestion and passage of rat BMSCs, the speed of MSCs in the process of digestion and passage was quicker than other bone marrow cells, as well as the characteristics of adherent speed, instead of density gradient centrifugation to separate and purify MSCs, and their morphological characteristics were observed. Cell growth and proliferation of two kinds of culture method were compared with the density gradient centrifugation separation. Alkaline phosphatase and oil red staining results were observed to verify BMSC differentiation capacity, to detect the cell surface markers, to validate immune properties and to test its purity.
RESULTS AND CONCLUSION: The whole bone marrow adherent culture method isolated and differentially subcultured rat BMSCs. Flow cytometry and osteogenic and adipogenic culture results displayed cytoimmunity property, purity and differential capacity, which have no significant difference compared with density gradient centrifugation. However, cell viability and reproductive activity are obviously elevated.

中图分类号: