中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (46): 8555-.doi: 10.3969/j.issn.1673-8225.2010.46.002

• 软骨组织构建 • 上一篇    下一篇

兔膝关节软骨细胞的分离培养及形态学特征

胡志俊1，2，胡波1，唐德志3，张有为3，王世伟2，王拥军1   

  1. 1上海中医药大学脊柱病研究所，上海市 200032；  上海中医药大学附属龙华医院，  2康复科，3骨伤科，上海市  200032
  • 出版日期:2010-11-12 发布日期:2010-11-12
  • 作者简介:胡志俊☆，男，1962年生，湖北省人，汉族， 2004年上海中医药大学毕业，博士，副教授，主要从事中医药对骨退行性变的康复研究。hzjz1062@163.com 并列第一作者：胡波★，男，1982年生，江西省南昌市人，汉族，2009年上海中医药大学毕业，硕士，主要从事骨关节疾病的治疗研究。 hubo585450@163.com
  • 基金资助:

    课题受上海市科委基础重点项目(08JC1418700)和上海市医学领军人才支持计划(05YLJ018)资助。

Isolation, culture and morphological characteristics of rabbit articular cartilage cells

Hu Zhi-jun 1, 2, Hu Bo1, Tang De-zhi3, Zhang You-wei3, Wang Shi-wei2, Wang Yong-jun1   

  1. 1Institute of Spine Disease, Shanghai University of Traditional Chinese Medicine, Shanghai  200032, China; 2Department of Rehabilitation, 3Department of Orthopaedics and Traumatology, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai  200032, China
  • Online:2010-11-12 Published:2010-11-12
  • About author:Hu Zhi-jun☆, Doctor, Associate professor, Institute of Spine Disease, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China; Department of Rehabilitation, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China hzjz1062@163.com Hu Bo, Master, Institute of Spine Disease, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China hubo585450@163.com
  • Supported by:

    the Key Program of Shanghai Science and Technology Commission, No. 08JC1418700*; Program for Outstanding Medical Leader in Shanghai, No. 05YLJ018*

PDF

456

被引次数

38

摘要：

背景：软骨细胞是软骨破坏过程中的靶细胞，也是分泌炎性细胞因子的主要细胞之一，体外分离培养骨关节炎软骨细胞存在难度。
目的：对兔膝关节软骨细胞进行分离和培养，观察兔软骨细胞的形态学特性。
方法：4周龄新西兰白兔采用机械-酶消化法，通过调整胰蛋白酶和Ⅱ型胶原酶的浓度及消化时间来获得较纯的关节软骨细胞，传代培养。通过倒置相差显微镜下观察细胞形态、绘制生长曲线、苏木精-伊红染色、阿利新蓝染色及Ⅱ型胶原免疫荧光染色对细胞进行鉴定。
结果与结论：原代培养的软骨细胞以多角形或三角形为主，传代3次后出现反分化。形态学、免疫荧光染色显示细胞培养3代以内可以保持表型的稳定，90%以上的软骨细胞维持多角形或三角形，核为圆形或椭圆形。苏木精-伊红阳性染色为细胞核呈紫蓝色，软骨细胞基质红染，胞浆及细胞周围有紫红色、阿利新蓝染色可见细胞浆和胞膜呈深蓝色、Ⅱ型胶原免疫荧光染色可见胞浆和胞膜清晰绿色荧光，细胞核区域未见明显绿色荧光。结果提示，实验成功建立了软骨细胞分离、培养体系，且3代以内90%以上的软骨细胞生长良好。

关键词: 兔, 关节软骨, 软骨细胞, 细胞分离, 细胞培养, 组织构建

Abstract:

BACKGROUND: Cartilage cells are target cells during cartilage damage, which are the main cells secreted inflammatory cytokines. It is difficult to isolate osteoarthritis cartilage cells.
OBJECTIVE: To isolate and culture rabbit articular cartilage cells and to observe the morphological characteristics of rabbit cartilage cells.
METHODS: Articular cartilage cells were obtained by mechanical-enzymatic digestion from the cartilage of 4-week-old New Zealand white rabbits. The cells were identified by phase-contrast microscope, growth curve, hematoxylin-eosin staining, Alician blue staining and type Ⅱ collagen immunofluorescence staining.
RESULTS AND CONCLUSION: The cultivation of cartilage cells presented with polygonal or triangle, and turned to dedifferentiation after third generation. Morphology and immunofluorescence staining showed that stable phenotype could be maintained within three generations. 90% cartilage cells maintained polygonal or triangle with circular or oval nuclei. Hematoxylin-eosin staining showed nucleus stained purple, cartilage matrix stained red, and surrounding cells or cytoplasm stained amaranth. Alician blue staining showed that cell plasma and membrane presented with dark blue. Type Ⅱ collagen immunofluorescence staining showed clear cytoplasm and cell membrane, which presented with green fluorescence, but there was not green fluorescence in the nucleus. Experimental results demonstrated that, a system that can isolate and culture cartilage cell is successful established. Over 90% cartilage cells grow well within three generations.

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