中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (28): 5136-5140.doi: 10.3969/j.issn.1673-8225.2011.28.002

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

多次胶原酶消化法培养小鼠椎间盘髓核细胞

张兴凯，曹  鹏，王  君，梁  裕，吴文坚   

  1. 上海交通大学医学院附属瑞金医院骨科 上海市伤骨科研究所，上海市  200025
  • 收稿日期:2011-02-20 修回日期:2011-05-10 出版日期:2011-07-09 发布日期:2011-07-09
  • 通讯作者: 曹鹏，博士，主任医师，上海交通大学医学院附属瑞金医院骨科 上海市伤骨科研究所，上海市 200025 caopeng8@sh163.net
  • 作者简介:张兴凯☆，男，1968年生，山东省济南市人，汉族，2004年上海交通大学医学院毕业，博士，副主任医师，主要从事脊柱外科研究。 zhangxk99@ 126.com
  • 基金资助:

    国家自然科学基金(81071502)，课题名称：缺氧诱导因子-1α(HIF-1α)对脊柱椎间盘发育及退变调控机制的研究。

In vitro culture of mouse nucleus pulposus cells of intervertebral disc by multiple enzymatic digestions

Zhang Xing-kai, Cao Peng, Wang Jun, Liang Yu, Wu Wen-jian   

  1. Department of Orthopedics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Institute of Traumatology and Orthopedics, Shanghai   200025, China
  • Received:2011-02-20 Revised:2011-05-10 Online:2011-07-09 Published:2011-07-09
  • Contact: Cao Peng, Doctor, Chief physician, Department of Orthopedics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Institute of Traumatology and Orthopedics, Shanghai 200025, China caopeng8@sh163. net
  • About author:Zhang Xing-kai☆, Doctor, Associate chief physician, Department of Orthopedics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Institute of Traumatology and Orthopedics, Shanghai 200025, China zhangxk99@126. com
  • Supported by:

    the National Nature Science Foundation of China, No. 81071502*

PDF

540

被引次数

4

摘要：

背景：椎间盘为无血运组织，椎间盘髓核细胞为分化终末细胞，细胞增殖能力较差，体外培养难度较大。
目的：探索小鼠椎间盘髓核细胞体外分离培养的方法。
方法：取小鼠椎间盘髓核组织，使用多次胶原酶消化的方法，分离培养髓核组织细胞，接种，传代，取第2代细胞，分别采用免疫细胞化学和RT-PCR方法检测椎间盘髓核细胞特征性分泌物Ⅱ型胶原和聚合蛋白的分泌量及mRNA的表达，并与软骨细胞，成骨细胞及成纤维细胞进行比较。
结果与结论：椎间盘髓核细胞贴壁后呈现软骨细胞的形态；Ⅱ型胶原和聚合蛋白染色均为阳性；Ⅱ型胶原和聚合蛋白mRNA表达与软骨细胞相同，与成纤维细胞和成骨细胞存在明显差别。说明多次胶原酶消化的方法可以获得大量纯净的椎间盘髓核细胞，性状稳定。

关键词: 椎间盘退变, 椎间盘髓核细胞, 体外培养, Ⅱ型胶原, 聚合蛋白

Abstract:

BACKGROUND: In vitro culture of nucleus pulposus cells of intervertebral disc is an important method to study degeneration of intervertebral disc. However, it is difficult to culture nucleus pulposus cells in vitro because the intervertebral disc is an avascular organ and nucleus pulposus cells poorly differentiate and proliferate.
OBJECTIVE: To establish the method for in vitro culture of nucleus pulposus cells of intervertebral disc, and provide a reliable tool for research of phenotype changes of nucleus pulposus cells in degenerative disc and disc cell transplantation for treatment of degenerative disc diseases.
METHODS: Mouse nucleus pulposus tissue was collected and repeatedly digested using collagenase. Cells were cultured and subcultured. The secretion of collagen Ⅱ and aggrecan of passage 2 cells were detected by immunohistochemistry and RT-PCR, The results were compared with other types of cells.
RESULTS AND CONCLUSION: Nucleus pulposus cells of intervertebral disc exhibit a chondrocyte-like shape after adhesion. Immunohistochemistry study showed that the cells were positive for collagen Ⅱ and aggrecan staining. RT-PCR study showed that secretions of collagen Ⅱ and aggrecan in cultured cells were equal to those of chondrocyes and had significant difference from those of osteoblasts and fibroblasts. Multiple enzymatic digestions of nucleus pulposus can release a large amount of pure nucleus pulposus cells which had stable phenotype and can settle basis for research and treatment of intervertebal disc diseases.

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