中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (1): 33-37.doi: 10.3969/j.issn.1673-8225.2010.01.008

• 干细胞培养与分化 • 上一篇    下一篇

人脐带间充质干细胞分离培养及成骨分化:原子力显微镜观察细胞膜表面的超微结构变化

孙国栋1，伍时县2，李志忠1   

  1. 1暨南大学附属第一医院骨科，广东省广州市  510632；
    2暨南大学生命科学技术学院化学系，广东省广州市  510632
  • 出版日期:2010-01-04 发布日期:2010-01-04
  • 通讯作者: 李志忠，主任医师，副教授，暨南大学附属第一医院骨科，广东省广州市 501630
  • 作者简介:孙国栋★，男，1982年生，山东省淄博市人，汉族，暨南大学在读硕士，医师，主要从事脊柱外科与骨组织工程的研究。 214404454@ qq.com 并列第一作者：伍时县，男，1983年生，广东省茂名市人，汉族，暨南大学在读硕士，主要从事生物纳米技术的研究。 740055486@qq.com
  • 基金资助:

    广州市科技局科技攻关计划项目(200623-E5191)

Isolation, culture and osteogenic differentiation of mesenchymal stem cells from human umbilical cord: Ultrastructure of cell membrane observed using atomic force microscope

Sun Guo-dong1, Wu Shi-xian2, Li Zhi-zhong1   

  1. 1Department of Orthopaedics, First Affiliated Hospital of Jinan University, Guangzhou   510632, Guangdong Province, China;
    2Department of Chemistry, College of Life Science and Technology, Jinan University, Guangzhou   510632, Guangdong Province, China
  • Online:2010-01-04 Published:2010-01-04
  • Contact: Li Zhi-zhong, Chief physician, Associate professor, Department of Orthopaedics, First Affiliated Hospital of Jinan University, Guangzhou 510632, Guangdong Province, China
  • About author:Wu Shi-xian★, Studying for master’s degree, Department of Chemistry, College of Life Science and Technology, Jinan University, Guangzhou 510632, Guangdong Province, China 740055486@qq.com Sun Guo-dong and Wu Shi-xian contributed equally to this article.
  • Supported by:

    the Science and Technology Research Plan Program of Technology Bureau of Guangzhou City, No. 200623-E5191*

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477

被引次数

12

摘要：

背景：目前，人们对间充质干细胞的观察研究多采用扫描电镜、透射电镜和倒置显微镜等，但是这几种方法对观察细胞膜超微结构及细胞骨架的研究则有不足之处。

目的：探讨并优化人脐带间充质干细胞体外获取及培养增殖的方法并鉴定；应用原子力显微镜观察其向成骨诱导过程中超微结构的变化。

方法：用酶消化法分离培养人脐带间充质干细胞，通过传代培养，扩增。体外向骨方向诱导分化，并通过碱性磷酸酶钙钴法染色、茜素红染色鉴定其分化能力。流式细胞仪检测人脐带间充质干细胞免疫表型；诱导过程中利用原子力显微镜的高空间分辨率从可视化的角度观察其在诱导前后细胞表面超微结构的变化。

结果与结论：采用酶消化法能有效分离纯化人脐带间充质干细胞。接种24 h，贴壁细胞形态多为长梭形，多边形或成纤维细胞样形态，大小均一。流式细胞仪分析第3代细胞均表达CD29、CD44 、CD105，不表达CD34、CD45和HLA-DR。经成骨诱导分化4周后，碱性磷酸酶染色呈强阳性，茜素红染色可见明显钙结节；通过原子力显微镜对分化前后的细胞骨架分析：未分化的干细胞其细胞骨架的微管突出不明显，分化后的细胞骨架其微管明显突出，并呈平行分布状态。

关键词: 原子力显微镜, 人脐带间充质干细胞, 成骨细胞, 诱导分化, 超微结构

Abstract:

BACKGROUND: Mesenchymal stem cells (MSCs) are commonly observed under the scanning electron microscope, transmission electron microscope and inverted microscope. However, above-mentioned observation methods have disadvantages on observing ultrastructure of cell membrane and cytoskeleton.

OBJECTIVE: To establish a more effective and appropriate method to isolate, culture and identification of human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro and to study the ultrastructure of osteogenic differentiation with the atomic force microscope.

METHODS: The hUCMSCs were isolated from human umbilical cord by digested with collagenase. After serial subcultivation in vitro, the stem cells were passaged, and osteogenic differentiation was determined by alkaline phosphatase calcium-cobalt staining and alizarin red staining. hUCMSCs immunophenotype was measured by Flow cytometry.The membrane surface ultrastructure of osteogenic differentiation was observed by Atomic Force Microscope before and after induction.

RESULTS AND CONCLUSION: The isolated hUCMSCs by digested with collagenase was efficient. After seeded 24 hours, the adherent cell showed spindle shape, polygonal shape and fibroblast-cell-like shape and the size of hUCMSCs was homogeneous. Flow cytometry analysis revealed that CD29, CD44, CD105 were highly expressed on the surface of passages 3 cells, but there was negative for CD34, CD45 and HLA-DR. These cells were high positive for alkaline phosphate staining and also showed significant calcium node using alizarin red staining after 4 weeks culture induction of osteogenic differentiation. Under atomic force microscopy, undifferentiated stem cells demonstrated insignificant microtubule protrution in a parallel distribution following analysis of cytoskeleton before and after differentiation.

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