不同月龄兔膝关节软骨单位原位及体外酶解消化形态学分析  

Histological analysis of chondrons isolated from age-related normal rabbit knee

作　　者：段王平[1] 孙振伟[1] 李琦[1] 赵昱[1] 卫小春[1] 

机构地区：[1]山西医科大学第二医院骨科,太原030001

出　　处：《中华骨科杂志》2016年第5期292-297,共6页Chinese Journal of Orthopaedics

基　　金：国家自然科学基金项目（31340010,31271033）;教育部高等学校博士学科点专项科研基金联合资助课题（20121417120004）;山西省基础研究计划项目青年科技研究基金项目（2013021036-3）;高等学校科技创新项目（20131105）;山西医科大学博士启动基金项目（03201114）

摘　　要：目的分析不同月龄兔膝关节软骨单位原位及体外酶解消化的形态学变化特点，探索软骨单位及细胞周基质在关节软骨退变机制中的作用。方法取新西兰白兔30只，按月龄分为3组：幼年组（2月龄）、中年组（8月龄）及老年组（31月龄），每组各10只。每组选5只兔，取其双侧膝关节股骨髁软骨进行原位组织切片形态学观察，包括HE、甲苯胺蓝及Ⅱ、Ⅵ型胶原免疫荧光染色。每组余下的5只兔，采用浓度为0．3g／L的Dispase酶和0．2g／L的Ⅱ型胶原酶联合搅拌消化3h的方法获取各组兔膝关节软骨单位，采用倒置显微镜及HE、VI型胶原免疫荧光染色等方法观察各组软骨单位体外组织形态学特点。结果随月龄增长，兔膝关节软骨组织中软骨细胞周基质界限逐渐清楚，软骨深层出现大量由细胞周基质包裹纵向排列2~4个软骨细胞的软骨单位形态。与单纯软骨细胞相比，体外酶解消化软骨单位细胞周基质成分明显、完整，保留了不同月龄软骨单位原位组织形态学特点，中、老年组软骨单位细胞周基质明显增厚，老年组由多个软骨细胞构成的软骨单位比例可达47％。结论随月龄增长，软骨单位及细胞周基质形态发生明显变化，提示细胞周基质在关节软骨成熟退变过程中维持软骨细胞微环境的作用。Objective To explore the histological properties of isolated chondrons and chondrocytes from rabbit knee cartilage, and to determine if these properties vary with age. Methods Three groups of rabbit knees were evaluated according to different age： （1） young （2 months, n=10）; （2） adult （8 months, n=10）; and （3） old （31 months, n=10）. The cartilage structure, proteo- glycan, collagen-2, and collagen-6 content were determined by light microscopic using hematoxylin-eosin （HE）, Toluidine Blue, and col-2, 6 staining. The chondrons were enzymatically isolated using 0.3 g/L dispase and 0.2 g/L collagenase-2 by shaking for 3 hours. The morphology and composition of isolated chondrons were observed by HE and collagen-6 immunostaining staining after overnight coverslip monolayer culture under a microscopy. Results The chondrocytes became sparser and the total content of proteoglycans and collagen-2 were decreased in the articular cartilage with age. Compared to the chondrocytes, the surrounding rim or capsule was more obvious in the isolated chondrons, and they exhibited obvious differences in shape. The cells within one cluster from different age groups were similar to the morphology observed in cartilage in situ. The aduh and old chondrons generally possessed a thicker pericellular matrix with more enclosed cells, and the chondrons contained more cells can reach 47%. Conclusion These findings further suggest that the properties of the chondrons and pericellular matrix have an important influence on the biomechanical microenvironment of the knee joint cartilage degeneration that occurs with age.

关 键 词：兔 膝关节 软骨细胞 胶原酶类 

分 类 号：R684[医药卫生—骨科学]