肽基精氨酸脱亚胺酶4在类风湿关节炎中的表达及意义-东南大学学报医学版

肽基精氨酸脱亚胺酶4在类风湿关节炎中的表达及意义

单位：1. 包头医学院第一附属医院风湿免疫科, 内蒙古包头 014010;
2. 内蒙古自治区自体免疫学重点实验室, 内蒙古包头 014010

分类号：R593.2

出版年·卷·期（页码）：2021·40·第二期（158-163）

摘要：

目的：分析类风湿关节炎（RA）患者肽基精氨酸脱亚胺酶4（PAD4）蛋白的表达水平与类风湿因子（RF）、抗环瓜氨酸肽（CCP）抗体及28个关节的疾病活动度评分（DAS28）的相关性，探讨PAD4对RA的诊断和治疗价值。方法：收集90例RA患者和76例健康对照者的血清，同时收集RA患者的临床资料，ELISA法检测PAD4的表达水平及RA患者血清中抗CCP抗体、RF-IgA、RF-IgG的表达水平。收集39例RA患者和22例健康对照者EDTA抗凝全血，采用淋巴细胞分离液分离外周血单个核细胞（PBMC），实时荧光定量PCR检测PAD4 mRNA的表达水平；特种蛋白仪检测89例RA患者血清中RF-IgM的水平。分离并培养RA患者PBMC，分别加入7.5、15、30、60 μmol·L^-1 PAD4抑制剂F-amidine，作用24、48及72 h后CCK-8检测PBMC增殖情况并确定最佳浓度和最佳作用时间；在最佳浓度和最佳作用时间时收集细胞采用流式细胞术检测PBMC细胞凋亡情况。结果：RA患者血清中PAD4的浓度为（5.94±0.37）ng·ml^-1，显著高于健康对照者的（2.30±0.18）ng·ml^-1，差异有统计学意义（P<0.05）。PAD4诊断RA的ROC曲线下面积（AUC）为0.875。RA患者PAD4的表达水平与RF-IgM呈正相关（r=0.22，P=0.039），抗CCP抗体阳性组患者PAD4的表达水平与抗CCP抗体水平呈正相关（r=0.258，P=0.033），而抗CCP抗体阴性组患者血清中也会有高水平的PAD4表达；PAD4表达水平与RF-IgA、RF-IgG和DAS28无相关性。PAD4抑制剂F-amidine作用RA患者PBMC的最佳作用时间是48 h，最佳作用浓度是15 μmol·L^-1。F-amidine可以增加PBMC晚期凋亡比例。RA患者全血中PAD4 mRNA的表达水平为7.23±9.57，显著高于健康对照者的1.00±0.00，差异有统计学意义（P<0.05），同时PAD4 mRNA的表达水平与抗CCP抗体呈正相关（r=0.390，P=0.023），但与RF及DAS28无相关性。结论：RA患者PAD4的表达水平可作为诊断RA的一个指标，在抗CCP抗体阴性的RA患者中也有73.7%的表达PAD4，检测PAD4的水平对其诊断价值更高；PAD4可以作为治疗RA的一个靶点。

Objective: To analyze the correlation between the expression of peptidyl arginine deiminase 4(PAD4) and the levels of rheumatoid factor(RF), anti-cyclic citrullinated peptide(CCP) antibody, the disease activity score using 28 joint counts(DAS28) in rheumatoid arthritis(RA) patients,and to explore the significance of PAD4 for the diagnosis and therapy of RA. Methods: The serum of 90 RA patients and 76 healthy controls were collected. At the same time, the clinical data of RA patients were collected. The expression level of PAD4,RF-IgA,RF-IgG and anti-CCP antibody in serum of RA patients and healthy controls was detected by enzyme linked immunosorbent assay(ELISA). In addition, EDTA anticoagulant blood of 39 RA patients and 22 healthy controls were collected, the peripheral blood mononuclear cells(PBMC) were separated by lymphocyte separation solution. The expression level of PAD4 mRNA of RA patients was detected by real-time fluorescence quantitative PCR, and the levels of rheumatoid factor in serum of RA patients were detected by BN Ⅱ System. The proliferation of PBMC was detected by CCK-8, and the apoptosis of PBMC was detected by flow cytometry. Results: The serum concentration of PAD4 in RA patients was(5.94±0.37) ng·ml^-1, which was higher than(2.30±0.18) ng·ml^-1 in healthy controls, the difference was statistically significant(P<0.05). The area under receiver operating characteristic(ROC)curve of PAD4 in diagnosing RA was 0.875. The expression level of PAD4 in RA patients was positively correlated with RF-IgM(r=0.22, P=0.039). The level of PAD4 in patients with positive anti-CCP antibody was positively correlated with anti-CCP antibody(r=0.258,P=0.033), moreover the level of PAD4 in patients with negative anti-CCP antibody were high. However, there was no correlation between level of PAD4 and RF-IgA, RF-IgG, DAS28. The best time and concentration of F-amidine, which was an inhibitor of PAD4, on PBMC were 48 h and 15 μmol·L^-1 respectively. F-amidine could increase the proportion of apoptosis in PBMC. The expression level of PAD4 mRNA in RA patients was 7.23±9.57, which was higher than 1.00±0.00 in healthy controls, the difference was statistically significant(P<0.05),which was positively correlated with anti-CCP antibody(r=0.390,P=0.023). However, there was no correlation between level of PAD4 mRNA and RF, DAS28. Conclusion: The expression level of PAD4 in RA patients can be used as an indicator for the diagnosis of RA, PAD4 is expressed in 73.7% RA patients with negative anti-CCP antibody. It is suggested that PAD4 can be used as the target of RA treatment.

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