兔膝关节骨性关节炎动物模型的复制与评价

Preparation and Determination of Rabbit Knee Osteoarthritis Model

目的 制作家兔膝关节骨性关节炎的模型,并通过观察软骨组织及软骨细胞退变情况进行检测.方法 取健康雄性家兔12只,体质量2.0～3.0 kg,随机分为2组,假手术组、模型组,每组6只,取右膝关节内侧纵切口切断前后交叉韧带和内侧副韧带并切除内侧半月板,逐层缝合伤口.术后每日肌肉注射青霉素2×105 U,连续7d,预防感染.每日强迫家兔活动60 min.12周后,处死全部动物,取完整膝关节作为实验标本.进行形态学分析,对2组分别进行细胞培养,观察兔软骨细胞的形态学特性.结果 组织形态学分析显示：假手术组藏红固绿染色显示正常组关节软骨表层光滑、平整,软骨细胞分布均匀,排序整齐,各层次清晰.模型组显示关节面软骨表层裂隙,有些出现局部缺损,软骨细胞数量明显减少,软骨层面染色不均匀,局部甚至出现失染.假手术组培养的软骨细胞大多呈梭形或短梭形,少部分呈多角形,细胞核为圆形或椭圆形,Ⅱ型胶原的表达明显;模型组培养的软骨细胞大多呈出现许多指状突起,呈多角形变化,核仁多染,细胞增殖速度明显减慢,Ⅱ型胶原的表达明显降低.结论 成功制作了兔膝关节骨性关节炎动物模型,并首次运用现代技术手段进行了分析,此模型操作简单,可重复性强,为进一步研究中医药防治膝关节骨性关节炎奠定了基础.

OBJECTIVE To establish rabbit knee osteoarthritis models and to observe the degeneration of cartilage tissue and cartilage cells.METHODS 12 healthy male rabbits,weighing 2.0～3.0 kg,were randomly divided into sham operation group and model group,6 in each group.Anterior cruciate ligament and medial collateral ligament were cut off in the right knee medial longitudinal incision and medial meniscus was resected,which was sutured by serial layers.2 × 105 U of penicillin were intramuscularly injected daily for 7 days to prevent inflammation.The rabbits were forced to exercise for 60 minutes a day; after 12 weeks,they were killed and the complete knees were taken out as experimental samples.They were processed with morphological analysis and the two groups were conducted in cell culture to observe the morphological characteristics of rabbit chondrocytes.RESULTS The histological analysis was that safranine fast green staining in sham operation group showed articular cartilage surface in normal group was smooth,flat,evenly distributed,aligned and clear of all layers; model group showed articular cartilage surface cracked,some part impaired,cartilage cells significantly reduced,cartilage level dyed uneven,and even the local unstained.Chondrocytes cultured in sham operation group were mostly fusiform or short spindle with a small part polygonal and nuclei circular or oval; expression of type Ⅱ collagen significantly reduced.CONCLUSION Rabbit knee osteoarthritis models are successfully established and analyzed first with modern techniques.The model operates simply and has a good repeatability,which lays a foundation for further study of TCM preventing and treating knee osteoarthritis.