沙利度胺对类风湿关节炎滑膜成纤维样细胞影响的体外研究

Effect of thalidomide on proliferation characteristics of fibroblast-like synovial cells in patients with rheumatoid arthritis in vitro

目的研究沙利度胺(Thalidomide)对类风湿关节炎(RA)滑膜成纤维样细胞(FLS)体外培养时的增生殖分化特性的影响。方法关节镜、关节活检针取RA患者滑膜组织,分离培养和鉴定FLS,MTT法和反转录-聚合酶链反应(RT-PCR)方法观察沙利度胺对FLS细胞存活分数(SF)和c-fos、COX-2mRNA表达。结果RA-FLS体外培养呈良性增生。沙利度胺对RA-FLS的SF值的抑制作用最强(P<0.05)。生理剂量的沙利度胺干预时的浓度与FLS的SF值呈负相关。RA-FLS的c-fos、环氧化酶2(COX-2)mRNA表达率高,加入沙利度胺共孵育3天后c-fos和COX-2mRNA的表达率均明显下降。结论培养和鉴定RA滑膜细胞,证实体外培养传代的RA-FLS为非恶性无限制增生。实验剂量下沙利度胺通过抑制c-fos、COX-2的表达、降低FLS增殖能力等不同途径,发挥对RA滑膜细胞的免疫调节作用。

Objective To investigate the proliferation characteristics of fibroblast-like synovial cells（FLS） in RA patients in vitro and the mechanism of the immunosuppressive effect of thalidomide on them. Methods Synovial specimens were obtained from patients with RA（6 cases） at the time of surgical arthrectomy, arthroscopy, blind needle biopsy, or pumping synovial fluid on knee. FLS was identified in vitro with histopathological dyeing, immunohistochemical analysis and fluorescence-activated cell sorting（ FCS） method for cell surface molecules respectively. Synoviocyte proliferation in RA was measured with MTT colorimetric assay and the survival fraction（SF）of FLS was evaluated according to the presence or absence of thalidomide. The expression of c-los and COX -2 mRNA was examined by semi-quantitative RT-PCR in FLS of RA patients. Results The identified synovial cells in patients with RA were FLS（Vimentin and Fibronectin expression positive）cul- tured with or without agents. The SF of FLS interfered by thalidomide was lower than con A（blank control without agents） in RA （P〈0.05） shown by MTT assay. The immunoregulation effects of physiological doses thalidomide was strong and demonstrated markedly dose-dependent inhibitory curses on FLS proliferation efficiency. It was detected by semi-quantitative RT-PCR that c-fos and COX-2 mRNA of RA-FLS expressed highly and decreased obviously after incubation with thalidomide. Conclusion There was no distinct activated unlimited viability in RA-FLS without being stimulated by proinflammatory cytokine in vitro. It is proved that thalidomide mightily moderates proliferation characters in RA. The therapeutic effect of thalidomide could be associated with its a- bilities to inhibit FLS excessive proliferation and differentiation, and it modulates c-fos and COX-2 mRANA expression distinctly in RA-FLS, without being induced by lymphocyte or cytokines.