Objective: To investigate the clinical significance of soluble Semaphorin 5A(Sema 5A) in patients of rheumatoid arthritis(RA) and the effect of Sema 5A on osteoclastogenesis in RAW264.7 cells. Methods: (1)Soluble Sema 5A was detected in the serum of 62 RA patients, 30 osteoarthritis(OA) patients and 48 healthy controls(HC) by enzyme-linked immunosorbent assay(ELISA).The relationships between serum Sema 5A and disease activity, radiographic severity and laboratory parameters were investigated in RA patients.(2)RAW264.7 cells were treated with different concentrations of Sema 5A(0,0.5,1,2.5,5 μg/ml).After 7 days, tartrate-resistant acid phosphate(TRAP) staining was performed. The mRNA levels of TRAP, cathepsin-K and matrix metallopeptidase 9(MMP-9) were tested using real-time polymerase chain reaction (RT-PCR).(3)Bone resorption area of dentine slides cultured with RAW264.7 cells was calculated after Sema 5A(5μg/ml) treatment. Results: (1)The serum Sema 5A in RA patients[(5.24±0.59)μg/L] was significantly higher than those in healthy controls[(2.93±0.34)μg/L,P<0.01] and OA patients[(2.68±0.47)μg/L,P<0.05]. The Sema 5A level in RA patients was positively correlated with disease activity score with 28 joint using C-reactive protein(DAS28-CRP), clinical disease activity index (CDAI),C-reactive protein(CRP) and sharp scores(P<0.05 or P<0.01).In addition,the serum Sema 5A in RA patients with positive anti-cyclic citrullinated peptide(CCP) antibody was significantly greater than that of anti-CCP antibody-negative patients(P<0.05).(2)After RAW264.7 cells were treated with Sema 5A, the number of TRAP positive osteoclasts increased according to the increase of Sema 5A concentration with maximal effect at 5 μg/ml. Meanwhile, Sema 5A promoted mRNA expression of TRAP,Cathepsin-K and MMP-9.(3)Bone resorption area increased when RAW264.7 cells were treated with Sema 5A(5 μg/ml). Conclusions: Serum Sema 5A is elevated in RA patients and correlated with disease activity and radiographic severity. Sema 5A promotes osteoclastogenesis of RAW264.7 cells.
目的： 研究类风湿关节炎（RA）患者血清神经轴突导向分子Semaphorin 5A（Sema 5A）表达及其对小鼠巨噬细胞系RAW264.7细胞向破骨细胞分化的作用。 方法： （1）选RA患者62例，骨关节炎（OA）患者30例，健康对照者48例，应用酶联免疫吸附（ELISA）法检测所有受试者血清Sema 5A水平。（2）以不同浓度Sema 5A（0、0.5、1、2.5、5 μg/ml）处理小鼠巨噬细胞系RAW264.7细胞，培养7 d后行抗酒石酸酸性磷酸酶（TRAP）染色，采用实时定量聚合酶链反应（PCR）检测TRAP、组织蛋白酶-K、基质金属蛋白酶-9 mRNA表达水平；（3）以5 μg/ml Sema 5A处理小鼠巨噬细胞系RAW264.7细胞7 d，培养基中预先加入薄骨片，显微镜下观察骨吸收陷窝个数。 结果： （1）RA患者血清Sema 5A水平显著高于健康对照者和OA患者[（5.24±0.59）μg/L比（2.93±0.34）μg/L比（2.68±0.47）μg/L，P<0.05）]；抗环瓜氨酸多肽（CCP）抗体阳性RA患者血清Sema 5A水平显著高于抗CCP抗体阴性者（P< 0.05）。Sema 5A水平与RA患者以C反应蛋白计算的28个关节疾病活动度（DAS28-CRP）、C反应蛋白、临床疾病活动指数（CDAI）、Sharp评分呈正相关（r值分别为0.273、0.519、0.448、0.292，P<0.05）；（2）随着Sema 5A浓度增加，TRAP染色阳性细胞数逐渐增多，5 μg/ml Sema 5A作用最强。Sema 5A能诱导TRAP、组织蛋白酶-K、基质金属蛋白酶-9 mRNA表达上调；（3）与空白比，5 μg/ml Sema 5A骨吸收陷窝数增多。 结论： RA患者血清Sema 5A水平显著升高，与患者的疾病活动度及骨破坏程度相关。Sema 5A可直接诱导小鼠巨噬细胞系RAW264.7细胞向破骨细胞分化。.
Keywords: Arthritis, rheumatoid; Osteoclasts; Semaphorin 5A.