小鼠滑膜成纤维细胞的原代培养  

Primary culture for synovial fibroblasts in mice

作　　者：赵进军[1] 胡子有[2] 欧阳晴晴 毋静[1] 陈玉姣[1] 杨敏[1] 

机构地区：[1]南方医科大学附属南方医院风湿免疫科,广州510515 [2]南方医科大学附属南方医院实验中心,广州510515

出　　处：《重庆医学》2016年第2期228-231,共4页Chongqing medicine

基　　金：国家自然科学基金资助项目(30972747;81172875)

摘　　要：目的利用C57BL／6小鼠探索出一种改良的小鼠滑膜成纤维细胞（SF）的原代培养方法，以方便类风湿关节炎（RA）关于滑膜炎的研究。方法收集小鼠髋关节周围滑膜，去除内含的“蛋黄样”物，将滑膜移入含0．5%Ⅳ型胶原酶的EP管中，剪碎；置入37℃摇箱中振荡消化60min，然后在涡轮振荡仪上振荡1．5min，常规培养。结果细胞培养1周左右。进行第一次传代；大约在培养10d后滑膜巨噬细胞的数量达到最高峰，然后逐渐减少；在第3次传代（15～20d）后，滑膜巨噬细胞基本消失。用Vimentin标记的细胞免疫荧光化学染色显示SF细胞纯度超过95％；用Vimentin和CD90．2标记的流式细胞术显示其纯度达到95％，用CD54标记其纯度大约为80％。结论利用C57BL／6小鼠探索出一种改良的小鼠SF的原代培养方法是一种简便易行的小鼠SF的原代培幕方法。Objective The primary culture of synovial fibroblasts is a convenient tool to study the pathology and physiology of synovial tissues. An improved method was constructed in this study by C57BL/6 mice to study the mechanism of rheumatoid arthritis（RA）. Methods The synovium around the hip joints were collected. Attention should be paid to eliminate the ＂egg-yolk＂ like yellow oval substance in the middle of the synovlum. The synovlum was transferred into a 1.5 mL Eppendorf tube containing 0.5 type IV eollagenase and cut into 1 mma blocks or so. The Eppendorf tube was placed in 37 ℃ Constant temperature orbital shaker incubator for 60 min. After digestion,the tube was placed on the Vortex for a high-speed oscillation for 1.5 minutes to guarantee the separation of cells. Results Within about 1 week,the first passage was performed by the trypsin digestion method. On day 10, the number of synovial macrophages reached the maximum and then decreased gradually. After the third generation （day 15 to 20）, the synovial macrophages generally disappeared. Vimentin was suitable for the immunoftuoreseence cytoehemical staining for the synovial fibroblasts. The cell purity was indicated as 〉95 %. The cytometric analysis indicated that purity of Vimentin and CD90.2- labelled cells was over 95%;the purity of CD54-1abelled ceils was 80% approximately. Conclusion It is a simple and effective method for primary culture of synovial fibroblasts in mice.

关 键 词：关节炎 类风湿 成纤维细胞 滑膜 细胞培养技术 小鼠 近交C57BL 

分 类 号：R593.22[医药卫生—内科学]