生长期兔髁突纤维软骨细胞和骨骺透明软骨细胞生物学特性的比较研究

目的 建立软骨细胞体外培养模型，研究纤维软骨和透明软骨的生物学差异。 方法 分离4周龄雌性兔双侧髁突软骨和膝关节骨骺软骨，采用胰酶和胶原酶联合消化的方法收集软骨细胞，分别进行体外培养并连续传代至P10。使用倒置显微镜观察细胞形态；采用阿利新蓝和Ⅱ型胶原免疫组化染色的方法分别对髁突纤维软骨细胞和骨骺透明软骨细胞进行鉴定；用细胞计数的方法绘制生长曲线；运用荧光定量PCR和Western印迹技术对软骨特异性指标Ⅰ型胶原、Ⅱ型胶原、Ⅹ型胶原、SOX9和Aggrecan进行检测，并使用SPSS 13.0软件包对数据进行统计学分析。 结果 倒置显微镜观察示，髁突纤维软骨细胞和骨骺透明软骨细胞形态均会随细胞传代而发生改变，且P2代以后改变明显；基因和蛋白水平的检测均证实P2代后软骨细胞去分化明显。阿利新蓝和Ⅱ型胶原免疫组化染色鉴定结果均为阳性，对照组为阴性。实时定量荧光PCR和Western印迹结果证明：在纤维软骨中，Ⅰ型胶原的表达量高于透明软骨；而Ⅱ型胶原、Ⅹ型胶原、SOX9和Aggrecan的表达量却明显低于透明软骨。 结论 使用本方法培养软骨细胞简单有效；髁突纤维软骨细胞和骨骺透明软骨细胞体外培养均存在去分化现象，且两者Ⅰ型胶原、Ⅱ型胶原、Ⅹ型胶原、SOX9和Aggrecan的表达存在显著差异，生物学特性并不相同。

A comparative study on the biological characteristics of fibrocartilage cells and hyaline cartilage cells of the growing rabbit

Objective To establish the model of chondrocyte cultured in vitro,and to study the biological differences between fibrocartilage and hyaline cartilage. Methods Condylar cartilage and epiphysis cartilage were aseptically dissected from 4 week old New Zealand white rabbit.Cells were obtained by enzyme digestion and cultured in vitro until the 10th passage.The fibrocartilage cells and hyaline cartilage cells were observed and analyzed by inverted microscope,alcian blue staining and immunohistochemical staining.Cells growth curves were made with the method of cell counting.Quantitative real time PCR and western blot technique were used to detect and analyze the expression of collagen Ⅰ,collagen Ⅱ,collagen Ⅹ,SOX9 and Aggrecan.One way ANOVA with SPSS13.0 software package was performed. Results With the increase of subcultures,both fibrocartilage cells and hyaline cartilage cells changed morphologically into fibroblast like cells,especially after P2.Detection of gene and protein level has demonstrated that the dedifferentiation of chondrocytes was obvious after P2 generation.The chondrocytes staining was positive in the slides of cells.Quantitative real time PCR and western blot both showed that the expressions of colagenⅠ,collagen Ⅱ,collagen Ⅹ,SOX9 and Aggrecan in fibrocartilage were significantly different from those in hyaline cartilage.The expression of colagenⅠin fibrocartilage was higher than that in hyaline cartilage while other expressions were lower. Conclusions It is a simple and effective way to culture chondrocytes in vitro in this study.Our research confirms that the tendency of dedifferentiation would occur in both fibrocartilage cells and hyaline cartilage cells.There are essential differences between fibrocartilage and hyaline cartilage in biological characteristics.