中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (11): 1973-1076.doi: 10.3969/j.issn.1673-8225.2011.11.018

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

大鼠椎间盘纤维环细胞的培养及鉴定

张坤木1, 2，陈少清1, 3，宋红梅2, 4，何  艳5，廖  军1, 3，洪  钰1, 4，王诗忠1, 2, 3   

  1. 1福建中医药大学康复医学研究所，福建省福州市 350003
    2福建中医药大学附属第二人民医院，福建省福州市 350003
    3国家中医药管理局中医康复技术三级实验室，福建省福州市350003
    4福建省运动功能康复重点实验室，福建省福州市 350003
    5福建医科大学生物化学与分子生物学实验室，福建省福州市350000
  • 收稿日期:2010-10-16 修回日期:2011-01-12 出版日期:2011-03-12 发布日期:2011-03-12
  • 通讯作者: 王诗忠，博士，教授、主任医师，博士生导师，福建中医药大学康复医学研究所，福建省福州市 350003；福建中医药大学附属第二人民医院，福建省福州市 350003；国家中医药管理局中医康复技术三级实验室，福建省福州市350003 fujiantuina@126.com
  • 作者简介:张坤木★，男，1984年生，福建省漳州市人，汉族，福建中医药大学在读硕士，主要从事脊柱康复相关工作。 fujiantuina@126.com
  • 基金资助:

    课题受国家自然科学基金(C190203)，卫生部科学研究基金(WKJ2008-2-48)，福建省科技厅国际合作项目(2009I0010)和陈可冀中西医结合发展基金(Ckj2008012)资助。

Culture and identification of rat annulus fibrosus cells

Zhang Kun-mu1, 2, Chen Shao-qing1, 3, Song Hong-mei2, 4, He Yan5, Liao Jun1, 3, Hong Yu1, 4, Wang Shi-zhong1, 2, 3   

  1. 1Institute of Rehabilitative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou  350003, Fujian Province, China
    2Second People’s Hospital, Fujian University of Traditional Chinese Medicine, Fuzhou  350003, Fujian Province, China
    3 TCM Rehabilitating Technique Level 3 Laboratory of State Administration of Traditional Chinese Medicine, Fuzhou  350003, Fujian Province, China
    4Fujian Movement Function Rehabilitation Key Laboratory, Fuzhou  350003, Fujian Province, China
    5Laboratory of Biochemistry and Molecular Biology, Fujian Medical University, Fuzhou  350000, Fujian Province, China
  • Received:2010-10-16 Revised:2011-01-12 Online:2011-03-12 Published:2011-03-12
  • Contact: Wang Shi-zhong, Doctor, Professor, Chief physician, Doctoral supervisor, Institute of Rehabilitative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350008, Fujian Province, China; Second People’s Hospital of Fujian University of Traditional Chinese Medicine, Fuzhou 350003, Fujian Province, China; TCM Rehabilitating Technique Level 3Laboratory of State Administration of Traditional Chinese Medicine, Fuzhou 350003, Fujian Province, China fujiantuina@126.com
  • About author:Zhang Kun-mu★, Studying for master’s degree, Institute of Rehabilitative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350008, Fujian Province, China; Second People’s Hospital of Fujian University of Traditional Chinese Medicine, Fuzhou 350003, Institute of Rehabilitative medicine Fujian Province, China fujiantuina@126.com
  • Supported by:

    the National Natural Science Foundation of China, No. C190203*; the Science Research Foundation of Ministry of Health, No. WKJ2008-2-48*; International Cooperation Project of Science and Technology Commission of Fujian Province, No. 2009I0010*; Development Fund of Chen Ke-ji Traditional and Western Medicine, No. Ckj2008012*

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569

被引次数

15

摘要：

背景：目前关于椎间盘纤维环细胞的培养方面还没有形成统一的标准，导致实验的操作性和重复性存在缺陷。
目的：通过对椎间盘纤维环细胞的体外培养，观察细胞的演变，以期为组织工程研究椎间盘纤维环提供方法和体外模型。
方法：采用酶消化法分离SD大鼠椎间盘纤维环细胞，进行单层培养，实验在原代培养中使用体积分15%胎牛血清的DMEM培养基，细胞传代培养一代后可换成体积分数10%胎牛血清的DMEM培养基。倒置相差显微镜观察其形态结构和生物学特性，通过甲苯胺蓝染色和免疫细胞化学染色等方法对其细胞表型进行鉴定。
结果与结论：培养的椎间盘纤维环细胞形态为圆形或梭形；原代细胞生长较慢，传代细胞生长增快；细胞具有甲苯胺蓝易染性；免疫细胞学方法检测表明纤维环细胞有Ⅰ，Ⅱ型胶原表达。结果证实，实验成功在体外培养了大鼠椎间盘纤维环细胞。

关键词: 生物学特性, 椎间盘, 纤维环, 细胞培养, 胶原

Abstract:

BACKGROUND: There is not a criterion for culture of annulus fibrosus cells, thus, there is deficiency in operability and repetitiveness.
OBJECTIVE: To establish the annulus cell lines and to observe their continuouschanges, in order to provide the methods and cell model for studying molecular biology in tissue engineering field.
METHODS: The annulus fibrosus cells were isolated with enzyme digestion and grew as monolayer. DMEM containing 15% fetal calf serum was used in the primary culture, which was replaced by DMEM containing 10% fetal calf serum after cell passage. Morphology, structure and biological characteristics of cells were detected by an inverted phase contrast microscope. The phenotype was identified with the toluiding blue staining and immunocytochemistry.
RESULTS AND CONCLUSION: The disc fibrinous ring cells were cultured in monolayer with the round or fusiformis morphology. The primary cells grew more slowly than the subculture cells. The cells displayed intense toluiding blue metachromasia; the immunohistochemical test revealed that the positive expression of type Ⅰ and Ⅱ collagen on the cells. The experiment successfully culture annulus fibrosus cells in vitro. 

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