中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (14): 2515-2519.doi: 10.3969/j.issn.1673-8225.2012.14.009

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

人骨髓间充质干细胞的体外培养、鉴定及成骨分化★

刘  伟1，刘  萌2，祝劲松1，郝红伟1，董宁征2，周海斌1    

  1. 1苏州大学附属第二医院骨科，江苏省苏州市215004；2苏州大学唐仲英血液学研究中心，江苏省苏州市  215004
  • 收稿日期:2012-01-10 修回日期:2012-01-28 出版日期:2012-04-01 发布日期:2012-04-01
  • 通讯作者: author: Zhou Hai-bin, Doctor, Associate chief physician, Department of Orthopedics, Second Affiliated Hospital of Soochow University, Suzhou 215004, Jiangsu Province, China zhouhaibins@yahoo.com.cn
  • 作者简介:刘伟★，男，1985年生，山东省滕州市人，汉族，苏州大学附属第二医院骨科在读硕士，主要从事骨与关节的基础与临床应用研究。aqqvvvv@126.com

In vitro culture, identification and osteogenic differentiation of human bone marrow mesenchymal stem cells

Liu Wei1, Liu Meng2, Zhu Jing-song1, Hao Hong-wei1, Dong Ning-zheng2, Zhou Hai-bin1   

  1. 1Department of Orthopedics, Second Affiliated Hospital of Soochow University, Suzhou 215004, Jiangsu Province, China; 2Tangzhongying Research Center of Hematology, Soochow University, Suzhou  215004, Jiangsu Province, China
  • Received:2012-01-10 Revised:2012-01-28 Online:2012-04-01 Published:2012-04-01
  • Contact: 周海斌，博士，副主任医师，苏州大学附属第二医院骨科，江苏省苏州市 215004 zhouhaibins@yahoo.com.cn
  • About author:Liu Wei★, Studying for master’s degree, Department of Orthopedics, Second Affiliated Hospital of Soochow University, Suzhou 215004, Jiangsu Province, China aqqvvvv@126.com

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715

被引次数

33

摘要：

背景：国内外对骨髓间充质干细胞的体外成骨诱导分化研究手段、测定指标均不够全面。
目的：建立并完善一整套人骨髓间充质干细胞的分离培养及鉴定方法，探讨其体外成骨分化能力。
方法：采用密度梯度离心法分离培养人骨髓间充质干细胞，流式细胞仪鉴定细胞表面表型。传至第3代时更换成骨诱导培养基进行成骨分化诱导。
结果与结论：人骨髓间充质干细胞生长旺盛，传代后增殖旺盛，第3代骨髓间充质干细胞表面表型CD44、CD73、CD90表达阳性，CD34表达阴性。诱导后的成骨细胞碱性磷酸酶活性增加，Gomori、Von kossa、茜素红染色均阳性。RT-PCR检测诱导后细胞有Ⅰ型胶原、碱性磷酸酶、骨钙素、骨唾液酸蛋白、骨桥蛋白及骨连接蛋白基因的表达，证明了人骨髓间充质干细胞成功向成骨方向分化。表明实验建立了一整套稳定、成熟的骨髓间充质干细胞分离、培养、扩增方案。

关键词: 骨髓间充质干细胞, 骨髓, 分离培养, 成骨细胞, 成骨分化

Abstract:

BACKGROUND: Research approaches and measurement index regarding osteogenic differentiation of bone marrow mesenchymal stem cells in vitro are not fully considered at home and abroad.
OBJECTIVE: To establish and consummate a set of methods of isolating, culturing and identifying human bone marrow-derived mesenchymal stem cells (hBMSCs) and investigate their osteogenic differentiation ability in vitro.
METHODS: hBMSCs were isolated using the method of density gradient centrifugation. The cell surface phenotypes of BMSCs were identified by flow cytometry. Passage 3 hBMSCs were induced by osteogenic culture medium for 2-3 weeks.
RESULTS AND CONCLUSION: The primary cultured hBMSCs grew and proliferated vigorously after subculture. Passage 3 hBMSCs were positive for CD44, CD73, CD90 and were negative for CD34. After osteogenic induction, alkaline phosphatase activity was increased and cells were stained positive by Gomori, Von kossa and alizarin red. RT-PCR results showed the gene expression of collagen Ⅰ, alkaline phosphatase, osteocalcin, bone sialoprotein, osteopotin and osteonectin. This confirms that the primary hBMSCs can differentiate into osteoblasts in vitro successfully. These findings suggest that a stable, mature method of isolating, culturing and amplifying hBMSCs has been established.

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