生长期兔髁突软骨细胞体外培养方法及其生物学特性研究

中国口腔颌面外科杂志 ›› 2014, Vol. 12 ›› Issue (1): 7-12.

生长期兔髁突软骨细胞体外培养方法及其生物学特性研究

黄林剑, 张秀丽, 李妙然, 李辉, 邓亚伟, 蔡协艺

上海交通大学医学院附属第九人民医院·口腔医学院口腔外科,上海市口腔医学重点实验室,上海 200011

收稿日期:2013-07-03 修回日期:2013-07-31 出版日期:2014-02-10 发布日期:2014-02-10
通讯作者: 蔡协艺,Tel:021-23271699-5705,E-mail: caixieyi27@126.com
基金资助:
国家自然科学基金(81200766,81070848); 上海市科学技术委员会资助项目(08DZ2271100,114119a3800,13140902702, 13XD1402300)

Study on behaviors of the growing rabbit condylar chondrocytes in vitro

HUANG Lin-jian, ZHANG Xiu-li, LI Miao-ran, LI Hui, DENG Ya-wei, CAI Xie-yi

Department of Oral Surgery, Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine; Shanghai Key Laboratory of Stomatology. Shanghai 200011, China

Received:2013-07-03 Revised:2013-07-31 Online:2014-02-10 Published:2014-02-10
Supported by:
Supported by National Natural Science Foundation of China (81200766, 81070848), and Research Fund of Science and Technology Committee of Shanghai Municipality (08DZ2271100, 114119a3800, 13140902702, 13XD1402300).

摘要/Abstract

摘要： 目的建立髁突软骨细胞体外培养模型,研究其生物学特性。方法分离4周龄健康新西兰大白兔双侧髁突软骨,采用胰酶和胶原酶联合消化方法收集4 h和12 h 2个时间点的髁突软骨细胞,连续体外培养并传代至P10。使用倒置显微镜观察细胞形态；采用甲苯胺蓝、阿利新蓝和免疫细胞化学染色对髁突软骨细胞进行鉴定；利用细胞计数绘制细胞生长曲线；通过Western 免疫印迹分析细胞Ⅱ型胶原、Ⅹ型胶原和SOX9在蛋白水平的表达情况。采用SPSS13.0软件包对Western 印迹条带灰度值进行统计学分析。结果兔髁突软骨细胞体积较大,呈纺锤形或多角形,铺路石样排列,细胞爬片染色结果为阳性。随着细胞传代“成纤维样”细胞比例逐渐加重,P2代之前的细胞形态差异较小。细胞生长曲线显示,兔髁突软骨细胞的体外培养符合经典的S形生长曲线。Western 印迹结果表明,4h和12 h所收髁突软骨细胞Ⅱ型胶原、Ⅹ型胶原和SOX9表达无显著差异。结论使用本方法培养兔髁突软骨细胞简单有效。髁突软骨细胞体外培养存在去分化现象,但P2代以内的髁突软骨细胞生物学特性相对稳定,适合用于后期实验研究,P3代以后的髁突软骨细胞逐渐丧失原有细胞的特性。

关键词: 兔, 髁突, 软骨细胞, 软骨细胞鉴定

Abstract: PURPOSE: To establish the growing rabbit condylar chondrocyte culture model in vitro, and to study its biological characteristics. METHODS: Condylar cartilage was aseptically dissected from the temporomandibular joint of 4-week old New Zealand white rabbit. Chondrocytes were obtained using enzyme digestion at the interval of 4 h and 12 h. Then they were cultured in vitro and subcultivated until the 10th passage. The chondrocytes were analysed by inverted microscope, toluidine blue staining, alcian blue staining and immunocytochemical staining. Condylar chondrocytes growth curve was made with the method of cell counting. Western blot was used to analyse the expression of collagen type Ⅱ, collagen type Ⅹ and SOX9. SPSS13.0 software package was used for statistical analysis. RESULTS: Condylar chondrocytes of rabbits were spindle-shape or polygon shape and displayed cobble-stone morphology. Following the increase of subcultures, the chondrocytes changed morphologically into fibroblast-like cells. The condylar chondrocytes staining was positive in the slides of cells, and the growth curve showed that condylar chondrocytes had a typical S shape.Western blot showed that the expression of collagen type Ⅱ, collagen type Ⅹ and SOX9 was not significantly different between condylar chondrocytes collected at 4 h and those at 12 h. CONCLUSIONS: It is a simple and effective way to culture condylar chondrocytes in vitro in this study. The results confirm that the tendency of dedifferentiation would occur after passage 3 which may lose its nature traits. The chondrocytes cultured from passage 0 to passage 2 in vitro are stable and suitable for subsequent research.

Key words: Rabbit, Condyle, Chondrocyte, Chondrocyte identification

黄林剑, 张秀丽, 李妙然, 李辉, 邓亚伟, 蔡协艺. 生长期兔髁突软骨细胞体外培养方法及其生物学特性研究[J]. 中国口腔颌面外科杂志, 2014, 12(1): 7-12.

HUANG Lin-jian, ZHANG Xiu-li, LI Miao-ran, LI Hui, DENG Ya-wei, CAI Xie-yi. Study on behaviors of the growing rabbit condylar chondrocytes in vitro[J]. China J Oral Maxillofac Surg, 2014, 12(1): 7-12.

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