中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (23): 4195-4198.doi: 10.3969/j.issn.1673-8225.2010.23.004

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

大鼠骨髓间充质干细胞体外增殖与多向分化的能力

许永涛1，李 锋2，鲁厚庚1   

  1. 1华中科技大学同济医学院附属荆州医院骨科，湖北省荆州市 434020；
    2华中科技大学同济医学院附属同济医院骨科，湖北省武汉市  430000
  • 出版日期:2010-06-04 发布日期:2010-06-04
  • 作者简介:许永涛，男，1976年生，湖北省仙桃市人，汉族，2009年同济医科大学毕业，博士，副主任医师，主要从事脊柱脊髓损伤与人工关节方面的研究。 xyt7512@medmail.com.cn

In vitro proliferation and multi-directional differentiation of rat bone marrow mesenchymal stem cells

Xu Yong-tao1, Li Feng2, Lu Hou-geng1   

  1. 1Department of Orthopaedics, Jingzhou Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Jingzhou   434020, Hubei Province, China;
    2Department of Orthopaedics, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan   430000, Hubei Province, China
  • Online:2010-06-04 Published:2010-06-04
  • About author:Xu Yong-tao, Doctor, Associate chief physician, Department of Orthopaedics, Jingzhou Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Jingzhou 434020, Hubei Province, China xyt7512@medmail. com.cn

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313

被引次数

17

摘要：

背景：生理条件下机体内多数骨髓间充质干细胞增殖并不明显，然而在一定刺激下可表现出旺盛的有丝分裂活动，具有很强的增殖倍增能力，且体外实验表明其具有多向分化潜能。
目的：进一步验证体外分离培养的大鼠骨髓间充质干细胞生长增殖与多向分化潜能。
方法：全骨髓法体外分离培养大鼠骨髓间充质干细胞，贴壁筛选法进行纯化，倒置相差显微镜下观察细胞形态及生长特征，MTT法绘制细胞生长曲线，免疫组化法对细胞表面干细胞标志CD44进行鉴定。取传至第4代细胞，分别用成骨、成软骨、成脂肪和成神经诱导剂予以培养，通过碱性磷酸酶染色、Ⅱ型胶原免疫组化染色、油红O染色、NeuN抗体免疫组化染色进行分化能力鉴定。
结果与结论：分离培养的细胞呈长梭形或多边形，细胞生长曲线呈S形，群体倍增时间约为31 h，免疫细胞化学染色后CD44呈阳性表达，CD34呈阴性。第4代骨髓间充质干细胞成骨诱导2周后出现钙盐沉积，成软骨诱导培养2周后Ⅱ型胶原检测呈阳性，成脂肪诱导培养2周后在细胞的胞浆内充满大量红色脂肪滴，成神经诱导6 h后细胞出现突起，类似神经元的轴突和树突纤维，NeuN免疫组化染色呈阳性。表明体外培养的大鼠骨髓间充质干细胞生长增殖能力旺盛，可向成骨细胞、软骨细胞、脂肪细胞、神经元样细胞方向分化。

关键词: 贴壁筛选法, 增殖, 诱导, 分化, 骨髓间充质干细胞

Abstract:

BACKGROUND: Proliferation of bone marrow mesenchymal stem cells is not clear under some physiological condition; however, mitosis occurs following stimulation to a certain degree. The bone marrow mesenchymal stem cells have a strong proliferation ability and multi-directional differentiation potency.
OBJECTIVE: To further investigate the in vitro proliferation and multi-directional differentiation of bone marrow mesenchymal stem cells.
METHODS: Bone marrow mesenchymal stem cells were separated using whole bone marrow culture method and purified using attachment method. Morphology and growth characteristics were observed under inverted phase contrast microscope and growth curve was drawn using MTT method. Immunohistochemical method was used to identify cell surface markers CD44 of stem cells. The fourth-passage cells were respectively cultured in osteogenic, adipogenic and neuro-differentiation induction medium, and confirmed using the alkaline phosphatase staining, type II collagen immunohistochemical staining, oil red O staining and NeuN antibody immunohistochemical staining.
RESULTS AND CONCLUSION: After isolation and culture, cells developed a spindle or polygonal appearance. Growth curve was S-shaped, and population doubling time was 31 hours. Immunohistochemical staining demonstrated that CD44 expression was positive, but CD34 was negative. Two weeks after induction, calcium salt mineralization was found in the bone marrow mesenchymal stem cells of passage 4. Two weeks after chondrogenic induction, type II collagen demonstrated a positive expression. Two weeks after fat-induced culture, a great quantity of red fat drops were observed in cytoplasm. Six hours after neural induction, cells displayed processes, which were similar to axon and dendrite fiber. NeuN immunohistochemical staining demonstrated a positive result. This suggested that in vitro cultured bone marrow mesenchymal stem cells had a strong proliferation and could differentiate into osteoblasts, chondrocytes, adipocytes, and neuronal-like cells.

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