两种方法分离培养类风湿性关节炎成纤维样滑膜细胞的比较

[摘要] 目的 比较体外分离培养成纤维样滑膜细胞（FLS）的方法。 方法 滑膜组织来自关节镜清洗术的活动期类风湿关节炎患者，分别采用消化酶培养法、组织块培养法分离培养FLS。采用倒置相差显微镜以及流式细胞术对第3～4代FLS进行鉴定。 结果 2种原代分离培养的方法均可成功培养出FLS。传代可使FLS达到形态学上的纯化要求，倒置相差显微镜观察均符合FLS的形态结构特征，流式细胞术检测示CD44以及CD90呈强阳性，CD55呈弱阳性。组织块法培养法FLS成功率较高，细胞游出时间较早，细胞损伤小。 结论 组织块培养法适合于FLS的培养，第3～4代细胞的数目、活性及纯度符合进一步实验的要求。

[关键词] 成纤维样滑膜细胞；细胞培养；分离培养；类风湿性关节炎

[中图分类号] R-33 [文献标识码] A [文章编号] 1674-4721（2012）07（c）-0013-03

Comparative of two methods for rheumatoid arthritis fibroblast-like synovial cells isolation

CAI Wenhong1 SUN Baodong1 ZHANG Baofeng2 CHENG Wenxiang3 YUE Ye3 HU Yiping3 LI Jinchao3 ZHANG Peng3

1.Department of Rheumatology, Shenzhen People′s Hospital in Guangdong Province, Shenzhen 518020, China; 2.Department of Nutrition, Shenzhen People′s Hospital in Guangdong Province, Shenzhen 518020, China; 3.Center for Translational Medicine R&D, Shenzhen Institute of Advanced Technology, Chinese Academy of Science, Guangdong Province, Shenzhen 518055, China

[Abstract] Objective To compare the isolated methods for fibroblast-like synoviocytes (FLS). Methods Synovium tissue obtained from patients with active rheumatoid arthritis by digestive enzyme culture and tissue culture. The 3～4 generation FLS were identified by morphology and flow cytometry. Results These two methods could culture FLS successfully, tissue culture method was better than the digestive enzyme culture method. FLS showed typical morphological characters under inverted phase contrast microscope. CD44 and CD90 were strong positive, and CD55 was weak positive in the FLS cell surface. Tissue culture method had higher successful rate with earlier and more quickly cell emigration from synovium tissue. Conclusion Tissue culture method is especially suitable for FLS culture in vitro from synovium tissue. And purity of FLS of the third or forth generation meet the requirement for molecular biology experiments.

[Key words] Fibroblast-like synoviocyte; Cell culture; Separate training; Rheumatoid arthritis

类风湿性关节炎（rheumatoid arthritis，RA）是一种慢性、炎性、系统性的自身免疫性疾病[1]。该疾病主要侵犯全身各处关节，呈多发性慢性增生性滑膜炎，可破坏关节软骨和关节囊[2-5]。而其病因至今仍未明确，为明确类风湿性关节炎的病因，发病机制以及寻找新的治疗方法，滑膜组织为重要的研究对象。

成纤维样滑膜细胞（fibroblast-1ike synoviocyte，FLS）是滑膜组织中的重要的组成细胞，可通过自分泌或旁分泌方式产生大量的炎性细胞因子及免疫反应介质，是参与RA发病的重要细胞[6]。建立FLS体外培养模型对于FLS功能的研究、进一步探讨RA的发病机制及研发新的RA的药物具有重要意义[7]。目前用于 FLS体外培养的滑膜多来自关节开放性手术、关节镜手术、从滑液中分离等方法[8]。本研究对比经典的消化酶培养法与组织块培养法进行的原代培养，比较获得体外培养滑膜组织FLS的最佳方法。为之后的其他研究型试验做基础。

1 材料与方法

1.1 实验材料