肿瘤坏死因子α对类风湿关节炎患者外周血单核细胞向破骨细胞分化的影响

Effect of tumor necrosis factor α on osteoclastogenesis of peripheral blood monocytes from rheumatoid arthritis patients

目的探讨肿瘤坏死因子α(TNF-α)对类风湿关节炎(RA)患者外周血单核细胞向破骨细胞分化的影响。方法采集RA患者外周血,密度梯度离心后经贴壁法纯化后获得单核细胞,分为A组、B组、C组、D组、E组、F组,分别加入核因子κB受体活化因子配体(RANKL)0 ng/ml+TNF-α0 ng/ml、RANKL 100 ng/ml+TNF-α0 ng/ml、RANKL 0 ng/ml+TNF-α5 ng/ml、RANKL 100 ng/ml+TNF-α2.5 ng/ml、RANKL 100 ng/ml+TNF-α5 ng/ml、RANKL 100 ng/ml+TNF-α10 ng/ml进行诱导培养。细胞培养14天后通过细胞形态学特征及抗酒石酸酸性磷酸酶(TRAP)染色鉴定所得细胞,并对TRAP染色阳性细胞进行计数。免疫印迹法检测TRAP染色阳性的各组细胞核因子κB受体活化因子(RANK)、活化T细胞核因子c1(NFATc1)蛋白的表达。结果 TRAP染色显示,A组、C组获得的细胞不具有典型破骨细胞特征,B组、D组、E组和F组的细胞符合破骨细胞TRAP染色特征。D组、E组和F组TRAP阳性细胞计数、RANK及NFATc1蛋白表达均高于B组(均P<0.05)。结论在缺乏RANKL的条件下,TNF-α不能独立诱导RA患者外周血单核细胞分化为破骨细胞,但存在RANKL时TNF-α能增加RANKL诱导生成的破骨细胞数量,这可能与其上调RANK、NFATc1蛋白的表达有关。

Objective To investigate the effect of tumor necrosis factor α（TNF-α） on the osteoclastogenesis of peripheral blood monocytes from rheumatoid arthritis（RA） patients. Methods Peripheral blood collected from RA patients were separated by density gradient centrifugation,then the monocytes were obtained after purification by adherence.The cells were divided into group A,group B,group C,group D,group E and group F,then were induced by receptor activator of nuclear factor-κB ligand（RANKL） 0 ng/ml＋TNF-α 0 ng/ml ,RANKL 100 ng/ml＋TNF-α 0 ng/ml,RANKL 0 ng/ml＋TNF-α 5 ng/ml,RANKL 100 ng/ml＋TNF-α 2.5 ng/ml,RANKL 100 ng/ml ＋TNF-α 5 ng/ml and RANKL 100 ng/ml＋TNF-α 10 ng/ml respectively.After 14 days of cell culture,the cells were identified by cell morphology and tartrate-resistant acid phosphatase（TRAP） staining,and then the TRAP-positive cells were counted.The protein expressions of receptor activator of nuclear factor-κB（RANK） and nuclear factor of activated T cells cytoplasmic 1（NFATc1） were detected by Western blotting in each group. Results TRAP staining showed that the typical osteoclast characteristics were not observed in the cells obtained from groups A and C,and osteoclasts with TRAP staining characteristics were observed in the cells of group B,group D,group E and group F.The counts of TRAP-positive cells,and the expressions of RANK and NFATc1 proteins in group D,group E and group F were higher than those in group B（all P 〈0.05）. Conclusion TNF-α can not induce monocyte differentiate into osteoclasts independently without RANKL in peripheral blood from RA patients.But TNF-α can increase the number of osteoclasts on the basis of RANKL probably by up-regulation of the expressions of RANK and NFATc1 proteins.