中国组织工程研究

中国组织工程研究 ›› 2014, Vol. 18 ›› Issue (23): 3639-3644.doi: 10.3969/j.issn.2095-4344.2014.23.006

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

幼兔髂骨穿刺抽取骨髓间充质干细胞分离培养鉴定：注意的细节与技术

张 聪1，刘洪美2，李庆伟1，陈国武1，梁 啸1，孟纯阳1   

  1. 1济宁医学院附属医院脊柱外科，山东省济宁市 272000；2济宁医学院病理教研室，山东省济宁市 272000
  • 修回日期:2014-03-20 出版日期:2014-06-04 发布日期:2014-06-04
  • 通讯作者: 孟纯阳，博士，主任医师，博士生导师，济宁医学院附属医院脊柱外科，山东省济宁市 272000
  • 作者简介:张聪，男，1989年生，河南省商丘市人，汉族，新乡医学院在读硕士，主要从事组织工程骨构建及应用研究。
  • 基金资助:

    山东省自然科学基金资助项目(ZR2010HM090)

Separation, culture and identification of rabbit bone marrow mesenchymal stem cells by iliac puncture: operation details and techniques

Zhang Cong1, Liu Hong-mei2, Li Qing-wei1, Chen Guo-wu1, Liang Xiao1, Meng Chun-yang1   

  1. 1Department of Spinal Surgery, Affiliated Hospital of Jining Medical University, Jining 272000, Shandong Province, China; 2Department of Pathology, Jining Medical University, Jining 272000, Shandong Province, China
  • Revised:2014-03-20 Online:2014-06-04 Published:2014-06-04
  • Contact: Meng Chun-yang, M.D., Chief physician, Doctoral supervisor, Department of Spinal Surgery, Affiliated Hospital of Jining Medical University, Jining 272000, Shandong Province, China
  • About author:Zhang Cong, Studying for master’s degree, Department of Spinal Surgery, Affiliated Hospital of Jining Medical University, Jining 272000, Shandong Province, China
  • Supported by:

     the Natural Science Foundation of Shandong Province, No. ZR2010HM090

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摘要：

背景：骨髓间充质干细胞被认为是构建组织工程骨修复骨与软骨缺损中较为常用的种子细胞，在其基本操作过程中注意常见问题并及时避免，对后期细胞学及组织工程学实验很有意义。

目的：通过作者实验操作过程中所遇问题的总结和分析，为初学者和科研人员提供可靠的骨髓间充质干细胞分离培养鉴定方法，减少操作过程中的人为失误和易犯问题。
方法：取16只幼年新西兰大白兔作为实验对象，进行髂骨穿刺抽取骨髓液。采用密度梯度离心法联合贴壁培养法体外筛选纯化细胞，并且通过倒置相差显微镜观察其形态学特点、生长曲线和流式细胞术鉴定骨髓间充质干细胞表型。

结果与结论：实验过程中前5只兔骨髓抽吸、骨髓间充质干细胞分离过程中遇到不同的问题和困难，经认真总结和分析，后11只兔骨髓抽吸、骨髓间充质干细胞分离均获成功，在细胞培养过程中未发现细菌污染和细胞老化，第3 代骨髓间充质干细胞高表达CD29、CD44抗原，而CD14、CD34抗原低表达，MTT测细胞生长曲线显示P3和P5增殖活性较高。尽管骨髓间充质干细胞分离培养鉴定技术已较为成熟，但是如果操作过程中不注意细节问题，也将会导致实验困难重重或失败。严格执行常规操作步骤可以得到纯度较高的骨髓间充质干细胞，提高成功率，为后续相关细胞实验和动物实验做好准备。

 


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程


全文链接：

关键词: 干细胞, 骨髓干细胞, 骨髓间充质干细胞, 种子细胞, 细胞培养, 表型鉴定, 兔, 山东省自然科学基金

Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells are considered as commonly used seed cells to construct tissue-engineered for repair of bone and cartilage defects. It is of great significance for cytology and tissue engineering experiments to study the common problems existing in the basic operation and how to avoid these problems in a timely manner.

OBJECTIVE: To summarize the common problems existing in the process of operation in order to provide reliable methods about separation, culture and identification of bone marrow mesenchymal stem cells for beginners and researchers. These can reduce or avoid some errors and problems during operation.
METHODS: Sixteen New Zealand white rabbits were selected as experiment objects, and bone marrow mesenchymal stem cells were separated from rabbits by iliac puncture, purified and augmented by using density gradient centrifugation combined with adherent culture method. Then cell morphology was observed by inverted phase contrast microscope, growth curve detected by MTT method and cell phenotype identified by flow cytometry.

RESULTS AND CONCLUSION: We encountered some problems in the process of separation and culture, when we operated the first five rabbits. After carefully summarizing and analysis of the reasons, the operation was successfully completed on the rest 11 rabbits. Bacteria pollution and cell aging were not found in the process of cell culture. What is more, the cells at passage 3 appeared with high-expression of CD29, and CD44, but low expression of CD14 and CD34. The cell growth curve showed that the proliferation activity of cells at passages 3 and 5 was higher than that at passage 10. Although the technology of separation, culture and identification of bone marrow mesenchymal stem cells is mature, the failure will be happen if we do not pay attention to the details of operation. By strictly carrying out normal operations, we can get high purity of bone marrow mesenchymal stem cells, which lays a good foundation for cell and animal experiments in the future.


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程


全文链接：

Key words: stem cells, mesenchymal stem cells, bone marrow, antigens, surface, cell proliferation

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