中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (46): 8569-8573.doi: 10.3969/j.issn.1673-8225.2011.46.005

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

原代培养差速贴壁法分离纯化大鼠腰椎间盘髓核细胞

孙浩林，李淳德   

  1. 北京大学第一医院骨科，北京市  100034
  • 收稿日期:2011-05-23 修回日期:2011-06-25 出版日期:2011-11-12 发布日期:2011-11-12
  • 通讯作者: 李淳德，主任医师，北京大学第一医院骨科，北京市 100034 lichunde@medmail.com.cn
  • 作者简介:孙浩林☆，男，1982年生，天津市人，汉族，2010年北京大学医学部毕业，博士，主治医师，主要从事脊柱外科研究。 sunhaolin5@medmail.com.cn
  • 基金资助:

    国家自然科学基金面上项目资助(81071504)。

Primary culture and purification of rat nucleus pulposus cells by anchorage velocity-dependent separation method

Sun Hao-lin, Li Chun-de   

  1. Department of Orthopedics, First Hospital, Peking University, Beijing 100034, China
  • Received:2011-05-23 Revised:2011-06-25 Online:2011-11-12 Published:2011-11-12
  • Contact: Li Chun-de, Chief physician, Department of Orthopedics, First Hospital, Peking University, Beijing 100034, China lichunde@medmail.com.cn
  • About author:Sun Hao-lin☆, Doctor, Attending physician, Department of Orthopedics, First Hospital, Peking University, Beijing 100034, China sunhaolin5@medmail.com.cn
  • Supported by:

    the National Natural Science Foundation of China, No. 81071504*

PDF

458

被引次数

23

摘要：

背景：髓核细胞在体外培养过程中存在表型丢失问题，包括Ⅱ型胶原、aggrecan、Sox-9等表达的下降，由类软骨细胞向类纤维样细胞转化。
目的：体外原代培养差速贴壁法分离纯化大鼠腰椎间盘髓核细胞。
方法：经胰蛋白酶、Ⅱ型胶原酶先后消化Wistar大鼠椎间盘髓核组织，第1，2代细胞传代时采用差速贴壁法分离纯化髓核细胞。
结果与结论：分离纯化后的第3代髓核细胞呈圆形或多角形，活力强，苏木精-伊红染色细胞核染成均一蓝黑色，胞浆呈现淡粉色；Ⅱ型胶原免疫组织化学染色阳性细胞比例为97%；aggrecan免疫组织化学染色阳性细胞比例为95%；扫描电镜可见细胞内有高尔基体、粗面内质网和游离核糖体，未见线粒体，可见少量板层小体；CCK-8生长曲线显示细胞经过2 d的生长潜伏期，3 d的指数生长期，进入生长停滞期。说明原代培养、两次差速贴壁法分离纯化的大鼠髓核细胞代谢旺盛、表型一致。

关键词: 髓核细胞, 原代培养, 差速贴壁法, 椎间盘, 大鼠

Abstract:

BACKGROUND: Nucleus pulposus cells have the problem of phenotype loss during in vitro culture, such as the decreasing expression of type II collagen, Aggrecan and Sox-9, leading to conversion of cartilage-like cells to fibroblast-like cells.
OBJECTIVE: To primarily culture rat nucleus pulposus cells purified by anchorage velocity-dependent separation method.
METHODS: The lumbar nucleus pulposus from Wistar rats were digested by trypsin and type II collagenase. The first and second passages of cells were isolated and purified by anchorage velocity-dependent separation method during subculture.
RESULTS AND CONCLUSION: The third passage of nucleus pulposus cells which were isolated and purified by anchorage velocity-dependent separation method appeared round or multi-angular with activity. Hematoxylin-eosin staining appeared homogeneous with blue nucleus and pink cytoplasm. The positive rate of type II collagen immunohistochemistry staining was 97% and rate of aggrecan immunohistochemistry staining was 95%. Transmission electron microscope results showed that there were many endoplasmic reaticulums, Golgi complexes, free ribosemes and few multilamellar bodies, but no chondriosome. The cell growth curve showed that after subculture, the third passage 3 of nucleus pulposus cells experienced 2 days of latent phase, 3 days of increased logarithmic phase, then went to plateau phase. Anchorage velocity-dependent separation method is a practical and effective method for purifying the primary cultured rat nucleus pulposus cells. The third passage of cells harvested through this method is active and homogeneous.

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