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

Study on behaviors of the growing rabbit condylar chondrocytes in vitro

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,4 h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。

PURPOSE： To establish the growing rabbit condylar chondrocyte cuhure 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 subcuhivated 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.