RA滑膜成纤维细胞的原代培养及鉴定

Primary Culture and Identification about Fibroblast-like Synoviocytes of RA

目的:改良体外分离、培养类风湿性关节炎滑膜成纤维细胞的方法,并进行鉴定。方法:取关节镜手术中获得RA患者滑膜组织进行机械分离、胶原酶消化后直接将所有消化产物置于细胞培养皿两次贴壁培养,差速消化法纯化成纤维细胞,倒置显微镜观察细胞形态、流式细胞术及免疫细胞化学的方法鉴定细胞纯度。结果:胶原酶消化后直接贴壁结合差速消化纯化法分离获得的原代滑膜细胞中呈梭形的成纤维样细胞占98%以上,细胞核呈椭圆形位于细胞中央,偶见少量圆形的滑膜巨噬细胞。流式细胞术显示98%以上的滑膜细胞具有vimeintin+CD68的成纤维细胞特征。免疫细胞化学提示滑膜细胞vimentin表达阳性、CD68不表达。结论:成功分离获得了纯度和活性很高的人滑膜成纤维样细胞,方法更简便,效率更高,为后续类风湿性关节炎滑膜侵袭机制的研究奠定了基础。

Objective：To improve the methods of isolation in vitro and culture fibroblast-like synoviocytes（FLSs） of rheumatoid arthritis（RA） and identification.Methods：The synovial tissue was taken from arthroscopic surgery of RA patients and mechanically separated.All products were digested by collagenase and placed in the cell culture dish to adherent culture twice.Fibroblasts were purified by differential digestion and observed cell morphology by inverted microscope.Furthermore,fibroblasts were identified cell purity by flow cytometry and immunocytochemistry.Results：In isolated primary synovial cells,FLSs were fusiform more than 98%.The nuclei were oval and located in the center of cells.Small amount circular synovial macrophages were occasionally observed in cells.Flow cytometry results showed positive expression of vimeintin and negative expression of CD68 more than 98% in synovial cells.Immunocytochemistry results showed that positive expression of vimentin and negative expression of CD68 in synovial cells.Conclusions：The method of isolation high purity and activity of human FLSs was successful.The method was simple and laid the foundation for the further study of the mechanism of synovial invasion in RA.