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目的观察类风湿关节炎(RA)滑膜组织中破骨细胞来源以及核因子κB受体活化子配体(RANKL)在诱导破骨细胞分化过程中的作用。方法取6例RA和6名正常关节的滑膜组织,用胶原酶消化法获得滑膜细胞,通过免疫磁珠法分选获得CD68+/-滑膜细胞。用外源性RANKL16μg/L、巨细胞集落刺激因子(M-CSF)25μg/L及地塞米松醋酸酯1×10-8mol/L诱导各组滑膜细胞分化。通过抗酒石酸酸性磷酸酶(TRAP)染色、降钙素受体(CTR)免疫荧光检测、骨吸收陷窝形成方法鉴定破骨细胞。并在RA滑膜CD68+细胞中加入0~8ng/ml梯度浓度的RANKL,观察不同浓度RANKL对RA滑膜CD68+细胞分化的影响。结果RA滑膜CD68+细胞在RANKL诱导14d后,RA滑膜CD68+细胞组出现CTR阳性、TRAP染色阳性细胞并有骨吸收陷窝形成。RA滑膜CD68+细胞在体外经RANKL诱导后分化形成的破骨细胞功能与RANKL剂量有关。结论RA滑膜组织中前体破骨细胞来源于滑膜CD68+细胞,在体外RANKL诱导下可以分化为成熟破骨细胞。RANKL体外诱导剂量影响RA滑膜CD68+细胞分化功能。
Objective To investigate the role of osteoclasts and RANKL in the induction of osteoclast differentiation in rheumatoid arthritis (RA) synovial tissue. Methods Six synovial tissues of RA and six normal joints were obtained and synovial cells were obtained by collagenase digestion. CD68 +/- synovial cells were obtained by immunomagnetic bead sorting. The differentiation of synoviocytes was induced by 16μg / L exogenous RANKL, 25μg / L M-CSF and 1 × 10-8mol / L dexamethasone acetate. Osteoclasts were identified by tartrate-resistant acid phosphatase (TRAP) staining, calcitonin receptor (CTR) immunofluorescence assay, and bone resorption lacuna formation. The RA synovium CD68 + cells were treated with RANKL at a concentration of 0 ~ 8ng / ml to observe the effect of RANKL on the differentiation of RA synovial CD68 + cells. Results On the 14th day after RANKL induction, RA synovial CD68 + cells showed positive CTR, TRAP positive cells and bone resorption lacuna formation. The function of osteoclasts differentiated by RA synovial CD68 + cells induced by RANKL in vitro was related to the dose of RANKL. Conclusion The precursor osteoclasts in RA synovial tissue originate from synovial CD68 + cells and can differentiate into mature osteoclasts under the induction of RANKL in vitro. In vitro induction dose of RANKL affected differentiation of RA synovial CD68 + cells.