中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (27): 4955-4959.doi: 10.3969/j.issn.1673-8225.2010.27.004

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

兔软骨细胞与骨髓基质细胞的平面共培养：两者培养比例筛选

孙 磊1，綦 惠1，陈 磊1，陈海燕1，江 健1，陶剑锋1，杰永生1，高新生1，Peter I. Lelkes 2   

  1. 1北京市创伤骨科研究所，北京积水潭医院，北京市100035；
    2School of Biomedical Engineering, Drexel University,  United States
  • 出版日期:2010-07-02 发布日期:2010-07-02
  • 作者简介:孙 磊，男，1966年生，黑龙江省牡丹江市人，汉族，1998年白求恩医科大学（现吉林大学白求恩医学院）毕业，博士，副研究员，主要从事软骨组织工程研究。 dr.sunlei2009@ gmail.com
  • 基金资助:

    北京市科技新星计划项目（合同编号：H013610310113）

Monolayer coculture of rabbit chondrocytes and bone marrow stromal cells: Screening of culture proportion

Sun Lei1, Qi Hui1, Chen Lei1, Chen Hai-yan1, Jiang Jian1, Tao Jian-feng1, Jie Yong-sheng1, Gao Xin-sheng1, Peter I. Lelkes2   

  1. 1Beijing Institute of Traumatology and Orthopaedics, Beijing Jishuitan Hospital, Beijing   100035, China;
    2School of Biomedical Engineering, Drexel University, United States
  • Online:2010-07-02 Published:2010-07-02
  • About author:Sun Lei, Doctor, Associate investigator, Beijing Institute of Traumatology and Orthopaedics, Beijing Jishuitan Hospital, Beijing 100035, China dr.sunlei2009@gmail.com
  • Supported by:

    the Science and Technology New Star Project of Beijing City, No. H013610310113*

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摘要：

背景：骨髓基质细胞一定条件下可以分化为软骨细胞，但目前还缺少将软骨与骨髓基质细胞平面共培养的深入探讨。
目的：拟明确骨髓基质细胞与软骨细胞共培养时比例的选择、细胞增殖活性、软骨特异性蛋白表达的规律，用于细胞移植的最佳时间和传代次数。
方法：软骨细胞与骨髓基质细胞的分离培养，传代后分为5组：单纯软骨细胞组；共培养组，软骨细胞与骨髓基质细胞7∶3，5∶5，3∶7组；以及单纯骨髓基质细胞组。传代至第4代(G4)，倒置相差显微镜观察各组细胞在不同传代时期的细胞形态，MTT实验检测细胞增殖活性，甲苯胺蓝与阿利新蓝染色，细胞免疫化学法检测Ⅱ型胶原的表达。
结果与结论：共培养各组在各个时期细胞表型正常。G1~G3代细胞增殖活跃，G4代细胞增殖能力下降。共培养各组甲苯胺蓝与阿利新蓝染色以及Ⅱ型胶原表达在G2和G3代均较高。综合各指标认为，以软骨细胞与骨髓基质细胞的比例为5∶5，3∶7组为最佳。软骨细胞与骨髓基质细胞平面共培养，保持了软骨细胞的形态和蛋白表达特性，如进行细胞移植，选取软骨细胞与骨髓基质细胞5∶5或3∶7的比例为最佳。

关键词: 软骨细胞, 骨髓基质细胞, 共培养, 蛋白聚糖, Ⅱ型胶原

Abstract:

BACKGROUND: Bone marrow stromal cells (BMSCs) can differentiate into chondrocytes under a certain condition, but there are lack of studies concerning monolayer coculture of chondrocytes and BMSCs at present.

OBJECTIVE: To identify percentage choice of coculture of BMSCs and chondrocytes, cell proliferation activity and cartilage specific protein expression rule, and to find the best time and subculture frequency during cell transplantation.

METHODS: Chondrocytes and BMSCs were isolated, cultured, and divided into 5 groups following passage: chondrocyte group, coculture (chondrocytes and BMSCs at 7: 3, 5: 5, 3: 7) groups, and BMSC group. At generation 4(G4), cell morphology was observed at different passages in each group under an inverted phase contrast microscope. MTT assay was performed for testing the cell viability. The proteoglycan expression was detected by toluidine blue and alcian blue staining. Type II collagen expression was determined by immunocytochemical method.

RESULTS AND CONCLUSION: Cells showed normal appearance in all the coculture groups. From G1-G3, cells had good viability, which was decreased at G4. Proteoglycan (detected by toluidine blue and alcian blue staining) and type II collagen expression were highly positively determined in the coculture groups during the second and third generations (G2 and G3). Synthetic each index showed that each index was best in the chondrocytes and BMSCs at 5: 5, 3: 7 groups. Results displayed that monolayer coculture of chondrocytes and BMSCs kept chondrocyte morphology and protein expression property. The proportion of chondrocytes and BMSCs at 5: 5 or 3: 7 was optimal for cell transplantation.

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