中国组织工程研究

中国组织工程研究 ›› 2014, Vol. 18 ›› Issue (1): 51-56.doi: 10.3969/j.issn.2095-4344.2014.01.009

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

全骨髓贴壁接触培养SD大鼠骨髓间充质干细胞

宫  宇，王鸿飞，夏海军   

  1. 大连医科大学附属第二医院骨外科，辽宁省大连市  116023
  • 修回日期:2013-10-13 出版日期:2014-01-01 发布日期:2014-01-01
  • 通讯作者: 王鸿飞，博士，教授，硕士生导师，大连医科大学附属第二医院骨外科，辽宁省大连市 116023
  • 作者简介:宫宇，男，1986年生，辽宁省铁岭市人，汉族，大连医科大学在读硕士，主要从事脊髓损伤修复与组织工程研究。
  • 基金资助:

    辽宁省科学技术计划项目基金(2011225013)，课题名称：NEP1-40与NT-3基因修饰的骨髓间充质干细胞联合应用对脊髓损伤修复作用的实验研究

Culturing bone marrow mesenchymal stem cells from Sprague-Dawley rats using whole bone marrow adherence method

Gong Yu, Wang Hong-fei, Xia Hai-jun   

  1. Department of Orthopedics, the Second Affiliated Hospital of Dalian Medical University, Dalian 116023, Liaoning Province, China
  • Revised:2013-10-13 Online:2014-01-01 Published:2014-01-01
  • Contact: Wang Hong-fei, M.D., Professor, Master’s supervisor, Department of Orthopedics, the Second Affiliated Hospital of Dalian Medical University, Dalian 116023, Liaoning Province, China
  • About author:Gong Yu, Studying for master’s degree, Department of Orthopedics, the Second Affiliated Hospital of Dalian Medical University, Dalian 116023, Liaoning Province, China
  • Supported by:

    the Science and Technology Plan Project of Liaoning Province, No. 2011225013

PDF

527

被引次数

13

摘要：

背景：体外分离培养出生长状态好、高纯度、增殖能力强和数量充足的大鼠骨髓间充质干细胞，是将其作为种子细胞用于组织和细胞移植的重要前提。
目的：建立简便、快速、有效的SD大鼠骨髓间充质干细胞体外分离培养方法，并观察其生物学特性。
方法：采用全骨髓法将SD大鼠双侧股骨和胫骨骨髓细胞进行体外分离培养，贴壁接种法进行细胞纯化、传代。观察细胞生长形态及特征，绘制细胞生长曲线，检测细胞表面标记物，采用体外诱导剂诱导细胞分别向成骨、成软骨、成脂方向分化。
结果与结论：全骨髓贴壁接种法分离培养的骨髓间充质干细胞生长旺盛、纯度高，细胞生长形态呈长梭形，极性排列，细胞生长呈S形生长曲线，群体倍增时间为29 h，细胞在连续传10代后仍具有较强的增殖能力。第3代骨髓间充质干细胞的表面标记物CD44、CD29、CD90均呈阳性表达，CD45、CD34、CD11b则呈阴性表达。第3代骨髓间充质干细胞分别经成骨、成软骨、成脂诱导剂诱导后，茜素红染色、碱性磷酸酶染色、von-kossa矿化结节染色、甲苯胺蓝染色和油红O染色均呈阳性。结果验证全骨髓贴壁接种法是一种简便可靠的体外分离培养方法，能获得纯度较高的骨髓间充质干细胞，经实验鉴定第3代骨髓间充质干细胞生物活性最佳，且具有多向诱导分化能力，适合作为后续实验的种子细胞。


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, SD大鼠, 分离, 培养, 贴壁法, 诱导分化

Abstract:

BACKGROUND: Tissue and cell implantation entails high-quality seed cells. In order to satisfy this requirement, it is crucial to produce adequate well-conditioned, high-purity and strong proliferation ability bone marrow-derived mesenchymal stem cells.
OBJECTIVE: To establish a simple, rapid and effective in vitro isolation and culture method of bone marrow- derived mesenchymal stem cells, and to define the biological features of bone marrow mesenchymal stem cells.
METHODS: Rat bone marrow mesenchymal stem cells were isolated from the bilateral tibial and femoral bones by the method of whole bone marrow, then purified and passaged by attachment method. The morphology and features of bone marrow mesenchymal stem cells were observed, the growth curve was drawn and the cell surface antigen was detected by flow cytometry. The bone marrow mesenchymal stem cells were induced to differentiate along the osteogenic, chondrogenic and adipogenic lineages.
RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells isolated by the whole bone marrow adherence method grew vigorously and were highly purified. The cultured cells were spindle-shaped. The growth curve was S-shaped and the population doubling time was 29 hours. The cells still maintained a strong proliferative capacity after they were passaged for 10 generations. The surface markers such as CD44, CD29, CD90 were positive, while CD45, CD34, CD11b were negative. At the third passage, bone marrow mesenchymal stem cells were induced to differentiate along the osteogenic, chondrogenic and adipogenic lineages, respectively. Following induction, Alizarin red staining, alkaline phosphatase staining, von-kossa mineralized nodules staining, toluidine blue staining, and oil red O staining were all positive. This shows that the whole bone marrow adherence method is a simple and reliable method for the in vitro isolation, culture and proliferation of bone marrow mesenchymal stem cells. Moreover, they have multi-lineage differentiation capacity under different inducers. The third passage bone marrow mesenchymal stem cells have the highest biological activity and can act as the ideal seed cells for subsequent experiments.


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: bone marrow, mesenchymal stem cells, rats, Sprague-Dawley, cells, cultured

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