人类风湿性关节炎成纤维样滑膜细胞培养及生物学特性研究

Isolation and characterization of human rheumatoid arthritis fibroblast-like synoviocytes

目的探讨人类风湿性关节炎(RA)成纤维样滑膜细胞(FLS)的分离及生物学特性。方法分别取4例RA患者膝关节滑膜组织、1例软骨瘤患者膝关节滑膜组织和1名健康人膝关节滑膜组织,采用组织块培养法培养FLS,使用相差显微镜观察细胞形态,流式细胞术(FCM)检测培养细胞的表面标志,酶联免疫吸附法定量检测RA第4代FLS细胞培养液上清液白细胞介素-1受体I型(IL-1RI)和白细胞介素1β(IL-1β)水平。结果组织块培养法成功培养出FLS。RA患者FLS细胞4～7d细胞数量明显增加,8～12d细胞可以长满瓶底。第3代以后细胞均质性最好,3～7代细胞稳定且增殖活跃,8代以后增长缓慢并逐渐老化。软骨瘤患者和健康人滑膜组织培养7d后才有散在单个FLS细胞爬出,大约16d后FLS细胞可以长满瓶底。FCM分析RA第4代FLSCD90+细胞的百分率为99.04%,CD3+为2.73%,CD3-CD19+为0.29%,CD3-CD16+CD56+为2.81%,CD14+为5.89%,CD55+为54.17%。RA第4代FLS细胞培养上清液IL-1RI浓度为(158.63±20.32)pg/ml,IL-1β为(4.67±0.82)pg/ml。结论组织块培养法能有效分离RA患者FLS,FLS具有高水平表达CD90、IL-1RI和IL-1β的特性。

Objective To isolate and characterize human rheumatoid arthritis （RA） fibroblast-like synoviocytes （FLSs）. Methods The synovial membrane tissues were obtained from 4 RA patients, 1 chondroma patient and 1 healthy subject and FLS were isolated by means of tissue culture. The cell morphology was observed by phase-contrast microscope and the cell surface markers were detected by flow cytometry. Results The FLSs were successfully cultured from the synovial membrane tissues with good cell homogeneity aider the third passage. The FLSs of the 3rd to 7th passages were stable and proliferated actively, followed by slow proliferation and aging since the 8th passage. Flow cytometry showed that the 4th-passage FLSs from the RA patients contained 99.04% CD90^＋ cells, 2.73% CD3^＋ cells, 0.29% CD3-CD19^＋ cells, 2.81% CD3^-CD16^＋CD56^＋ cells, 5.89% CD14^＋ ceils, and 54.17% CD55^＋ cells. The presence ofinterleukin-1 receptor type Ⅰ （IL-1RI, 158.63＋20.32 pg/ml） and IL-1β （4.67±0.82 pg/ml） were detected in the cell culture supernatant of the 4th-passage FLSs from the RA patients by enzyme-linked immunosorbent assay ELISA. Conclusion FLSs from RA patients can be effectively culture by means of tissue culture, and the cultured FLSs show high expressions of CD90, IL-1RI and IL-1β.