中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (2): 218-222.doi: 10.3969/j.issn.1673-8225.2010.02.007

• 软骨组织构建 cartilage tissue construction • 上一篇    下一篇

骨髓间充质干细胞向纤维软骨细胞表型的诱导分化

蔡贵泉，崔一民，陈晓东   

  1. 上海交通大学医学院附属新华医院骨科，上海市200092
  • 出版日期:2010-01-08 发布日期:2010-01-08
  • 通讯作者: 陈晓东，博士，教授，主任医师，博士生导师，上海交通大学医学院附属新华医院骨科，上海市 200092 chenxdmd@163.com
  • 作者简介:蔡贵泉★，男，1981年生，广东省汕头市人，汉族，上海交通大学医学院附属新华医院骨科在读硕士，主要从事半月板组织工程的研究。 caiguiquan99@sina.com
  • 基金资助:

    上海市教育委员会科研创新项目(08YZ37)。

Differentiation of bone marrow mesenchymal stem cells into fibrochondrocyte phenotype

Cai Gui-quan, Cui Yi-min, Chen Xiao-dong   

  1. Department of Orthopaedics, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai  200092, China
  • Online:2010-01-08 Published:2010-01-08
  • Contact: Chen Xiao-dong, Doctor, Professor, Doctoral supervisor, Department of Orthopaedics, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China chenxdmd@163.com
  • About author:Cai Gui-quan★, Studying for master’s degree, Department of Orthopaedics, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China caiguiquan99@sina.com
  • Supported by:

    Scientific Research Innovation Project of Shanghai Education Committee, No.08YZ37*

PDF

451

被引次数

22

摘要：

背景：半月板损伤后自行修复能力有限，组织工程技术为半月板损伤后修复开辟了一条新的途径。骨髓间充质干细胞具有多向分化潜能，有可能成为半月板组织工程理想的种子细胞。
目的：观察体外培养的猪骨髓间充质干细胞在诱导培养液作用下向纤维软骨细胞表型分化的可行性。
方法：采用全骨髓培养法分离培养猪骨髓间充质干细胞。取第3代的骨髓间充质干细胞，实验组以2.0×104/cm2细胞密度接种于24孔板，在含有转化生长因子β1、胰岛素样生长因子Ⅰ、地塞米松及抗坏血酸的DMEM-LG完全培养液中诱导分化，对照组以2.0×104/cm2细胞密度接种于24孔板，加入不含诱导因子的DMEM-LG完全培养液。在诱导第7，14，21天分别行甲苯胺蓝染色、免疫细胞化学染色检测其分化结果。
结果与结论： ①第2代骨髓间充质干细胞的群体倍增时间最短约为2 d，第4代以后相对延长，为5~9 d。②诱导后的骨髓间充质干细胞由梭形向多角形、多边形转变。③实验组可见胞浆呈明显的紫蓝色，尤其细胞密集生长的区域蓝染较深，染色强度随诱导时间延长而增强。对照组的骨髓间充质干细胞只有核蓝染。④实验组和对照组Ⅰ型胶原免疫细胞化学染色均阳性，各期染色无明显差异。对照组未见Ⅱ型胶原表达，实验组诱导14 d后可见Ⅱ型胶原能稳定的表达。提示骨髓间充质干细胞在体外诱导培养下可分泌纤维软骨细胞特征性细胞外基质，作为半月板组织工程种子细胞来源可行。

关键词: 骨髓间充质干细胞, 纤维软骨细胞, 诱导, 分化, 软骨组织工程

Abstract:

BACKGROUND: The meniscus has limited ability in repairing itself after being injured. However, tissue engineering provides a new way to meniscus repair after injury. Bone marrow mesenchymal stem cells (BMSCs), which possess the potential of multi-directional differentiations, can be ideal seed cells in meniscus tissue engineering.
OBJECTIVE: To investigate the feasibility of differentiation of in vitro cultured porcine BMSCs into fibrochondrocyte phenotypes in inductive medium.
METHODS: BMSCs were isolated with whole bone marrow culture method. Then, BMSCs of the third passage were digested and incubated in a medium containing transforming growth factor-β1, insulin-like growth factor-Ⅰ, dexamethasone and ascorbic acid in a 24-well plate at a density of 2.0×104/cm2 in the experimental group. While in the control group, the DMEM-LG complete culture medium containing no inductive factor were used instead. At day 7, 14 and 21 after induction respectively, Toluidine blue staining and immunocytochemical staining were performed to detect differentiation.
MAIN OUTCOME MEASURES: ①Population double time (PDT) of BMSCs; ②Morphological changes of BMSCs under light microscope; ③Proteoglycan expression; ④Collagen type Ⅰand type Ⅱ expression.
RESULTS AND CONCLUSION: ①The PDT of the second passage BMSCs was 2 days, which was the shortest. The PDT prolonged relatively after the fourth passage, which were 5 to 9 days. ②The BMSCs changed from a spindle-like appearance into a polygonal shape after induction. ③In the experimental group, toluidine blue staining resulted in hyacinthine-stained cytoplasm and the blue was even deeper in the area where cells were dense; The degree of staining increased with the increasing induction time. While in the control group, only nucleus of BMSCs were stained blue. ④Collagen type Ⅰ immunocytochemical staining was positive in both the experimental and the control group and there was no difference of significance between various induction time. No collagen type Ⅱwas seen expressed in the control group, while in the experimental group it was seen to be expressed steadily after 14 days of induction. It is indicated tlat BMSCs can be induced to synthesize fibrochondrocyte-characterized extracellular matrixes in vitro, which suggests that BMSCs are available as seed cells in meniscus tissue engineering.
 

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