骨性关节炎软骨下骨成骨细胞的分离培养与增殖活性的观察

Primary culture and proliferation activity identification of rabbits subchondral bone cell

目的探讨骨性关节炎软骨下骨成骨细胞的分离、培养、鉴定方法及生长特性。方法复制改良Hulth兔膝关节不稳模型；采用I型胶原酶消化联合组织块贴附法获得软骨下骨成骨细胞；应用倒置显微镜、I型胶原免疫化学染色以及瑞氏一姬姆萨染色来进行形态学观察和生物学鉴定；利用软骨细胞与滑膜细胞分别于软骨下骨成骨细胞共培养，应用四甲基偶氮唑蓝检测软骨下骨成骨细胞的增殖活性；检测细胞的I型胶原在基因水平的表达。结果I型胶原酶消化后在组织块贴附第11天有细胞开始从组织块周围爬出；I型胶原酶免疫化学染色后可见胞浆内出现黄褐色颗粒，为阳性反应。瑞氏-姬姆萨染色显示细胞染成蓝紫色，胞核饱满，核仁清晰；四甲基偶氮唑蓝检测显示在3d时，滑膜细胞和软骨细胞对于软骨下骨成骨细胞的增殖有着明显的促进作用，在第6、10、14天时这种促进作用变得不明显，而软骨下骨成骨细胞的增殖活性超过2个共培养组。I型胶原表达水平检测显示在第10天时，软骨细胞对于软骨下骨成骨细胞分泌I型胶原的促进作用最强，而滑膜细胞对其的促进作用不明显。结论酶消化联合组织块贴附法可获得理想的软骨下骨成骨细胞；利用I型胶原酶免疫化学染色鉴别软骨下骨成骨细胞方法简便易行；利用软骨下骨成骨细胞与软骨细胞和滑膜细胞共培养的体系适用于骨性关节炎（OA）微环境的研究，且该体系可以模拟类OA软骨下骨成骨细胞、滑膜细胞和软骨细胞相互影响作用的进程。

Objective To study the method of cell isolation, primary culture and identification of subehondral bone cell of osteoarthritis（OA） rabbits. Methods The rabbit instable knee joint models were made by modified Hulth modeling method. The osteoblasts were harvested from the subchondral bone of rabbits by collage- nase and tissue explants attachment. The morphology observation and biological identification were performed by inverted microscope and immunocytochemistry staining, respectively. The proliferative activity of cells were detected by MTT and the expression of I-collagen at gene level was detected. Results The cells started to appeared on the 1 lth day after the attachment. The cells form were fusiformis and triangle, the nucleolus were clear. The cultured cells had typical osteoblast morphological characteristics. The cells obtained from subchondral bone of rabbits were identified to be osteoblast by immunocytochemistry staining. The proliferative activity of cells were equably proliferation which detected by MTT. Conclusion The modified method provides better way to obtain ideal subchondral osteoblast and the co-culture method is suitable for the study of OA microenvironment, which can simulate interactions of the subchondral osteoblast, synovial cells and chondrocyte.