正常关节和骨关节炎软骨细胞凋亡和超微结构的研究

Changes in Ultrastructure Aspects and Apoptosis of Rabbit Normal and Osteoarthritic Chondrocytes

目的探讨正常关节和骨关节炎(OA)软骨细胞凋亡和超微结构的变化,以及正常关节和OA关节软骨细胞体外培养的凋亡来分析骨关节炎可能的发病机制。方法胡氏造模法制作新西兰大白兔OA模型,3周后切取关节软骨,末端原位标记染色法分析正常关节软骨,骨关节炎软骨的细胞凋亡。分离关节软骨细胞体外培养,然后通过钙黄绿素和双FITC-AI双染色,分析软骨细胞的活力和凋亡情况,同时切取关节软骨细胞行透射电镜分析软骨细胞中一些超微结构的变化。结果发现OA组软骨中凋亡细胞(14.32±3.17)比正常组(4.54±1.17)明显增多,两组间比较差异有统计学意义(t'=15.85,P<0.05);而体外培养的软骨细胞中,OA组分离的软骨细胞存在大量凋亡细胞(83.63±20.11)和正常组(91.45±4.70)比较,差异有统计学意义(t'=2.07,P<0.05)。OA组部分软骨细胞的活力明显增强,OA组为79.45±3.60,正常组为75.60±5.33(t=3.28,P<0.01)。在超微结构的研究中,发现大量软骨细胞内的超微结构,有明显的变化,特别是线粒体在正常组为7.3±2.3,而OA为3.4±1.7,明显减少,比较差异有统计学意义。OA组染色质边缘化,空泡化和分布异常也非常明显。结论在OA模型中,存在软骨细胞大量凋亡现象,通过超微结构现象分析凋亡细胞增多同时存在染色质的减少和分布异常,以及一些细胞小体的减少,可能和软骨细胞能量过度代谢和线粒体的减少等有密切的关系。

Objective To compare the ultrastructure changes and apoptosis of chondrocytes in the normal and osteoarthritis articular cartilage in order to investigated the possible pathogenesis of osteoarthritis. Methods New Zealand rabbits, Used Hu＇s osteoarthritis model, detected the apoptosis of chondrocytes both in normal and model articular cartilage and isolated from normal and osteoarthritic ar- ticular cartilage cultured in vitro. The apoptosis of articular cartilage detected byTUNEL, Cultured chondrocytes were stained with calcein and Annexin V -FITC/PI to analyzed the viability and apoptosis. Transmission electron microscope（TEM） analyzed the changes of ultra- structure of chondracyte in articular cartilage. Results The results showed that apoptosis of cartilage in osteoarthritis group（ 14.32±3.17）was increased much more than in normal group（4.54 ± 1.17） ,it had significantly different between the two groups （t＇ = 15.85, P 〈 0.05 ）. The apoptotic cells stained with Annexin V - FITC/PI in osteoarthritis group （ 83.63 ± 20.11 ） was higher than in normal group （91.45 ± 4.70）, it had significantly different between the two groups （ t ＇ = 2.07, P 〈 0.05 ）. The activity of chondrocytes stained with calcein in osteoarthritis group （79.45 ± 3.60） was stronger than normal group （75.60±5.33） , it had significantly different between the two groups （t = 3.28, P 〈 O. 01 ）. On the uhrastructural study, we found that there were significant changes in the ultrastructure of carti- lage ceils, especially the mitochondria significantly reduced in osteoarthritis group （3.4±1. 7 ） compared to normal group （7.3±2.3 ） , chromatin marginalization, vacuolization and abnormal distribution could be seen in osteoarthritis group obviously. Conclusion Apoptosis of chondrocyte was increased in osteoarthritis accompanied the changes of ultrastruetural and mitochondria reduce maybe related to abnor- mal energerv metabolism in chondrocyte.