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目的探讨类风湿关节炎(rheumatoid arthritis,RA)滑膜细胞中表达核因子κB受体活化子配体(RANKL)的细胞以及甲氨蝶呤(MTX)对其表达的影响。方法收集原代RA及正常滑膜细胞,用免疫磁珠法筛选滑膜CD68+和CD68-细胞,CD44免疫组织化学染色观察以及RANKL免疫荧光检测;细胞体外培养并在培养液中加入最终浓度为1mg/L的MTX,用双夹心法酶联免疫吸附试验(ELISA)检测培养液中RANKL含量以及评估每一细胞RANKL表达量。结果在滑膜细胞中,CD68+滑膜细胞为巨噬样细胞,CD44、RANKL染色阴性;CD68-滑膜细胞为纤维样细胞,CD44、RANKL染色阳性。RA滑膜CD44+/CD68-纤维样细胞RANKL蛋白表达量显著增高,MTX对其RANKL表达有抑制作用(P<0.01)。正常滑膜纤维样细胞低表达RANKL,MTX对其RANKL表达抑制作用来自对细胞增殖的抑制。结论滑膜组织中有RANKL表达,这些细胞为CD44+/CD68-纤维样细胞,RA滑膜纤维样细胞RANKL分泌增高并受到MTX抑制。MTX可通过抑制RANKL减少关节周围骨破坏作用。
Objective To investigate the effect of methotrexate (MTX) on the expression of RANKL in rheumatoid arthritis (RA) synoviocytes. Methods The primary RA and normal synovial cells were collected and the synovial CD68 + and CD68- cells were screened by immunomagnetic beads method. The CD44 immunohistochemical staining and RANKL immunofluorescence assay were performed. The cells were cultured in vitro and the final concentration was 1 mg / L of MTX. The content of RANKL in culture solution was detected by double sandwich enzyme-linked immunosorbent assay (ELISA) and the expression of RANKL in each cell was evaluated. Results In synovial cells, CD68 + synoviocytes were macrophage-like cells, with CD44 and RANKL staining negative. CD68- synovial cells were fibroblast-like cells with CD44 and RANKL staining positive. RANKL protein expression in RA synovium CD44 + / CD68-fiber-like cells was significantly increased, MTX inhibited the expression of RANKL (P <0.01). Normal synovial fibroblast-like cells expressed low levels of RANKL, and MTX inhibited the expression of RANKL from inhibition of cell proliferation. Conclusions The expression of RANKL in synovial tissue is CD44 + / CD68- fiber-like cells. RANKL secretion of RA synovial fibroblasts is increased and inhibited by MTX. MTX can reduce the destruction of bone around the joint by inhibiting RANKL.