中性粒细胞胞外诱捕网在抗磷脂综合征中的检测及形成机制的初步研究  

The detection and preliminary study on the formation mechanism of neutrophil extracellular traps in antiphospholipid syndrome

作　　者：解鸿翔 许国莹 张巧云 成思 朱里洁 孙涛 靳仙宝 Xie Hongxiang;Xu Guoying;Zhang Qiaoyun;Cheng Si;Zhu Lijie;Sun Tao;Jin Xianbao(Department of Clinical Laboratory, the First Affiliated Hospital of Shandong First Medical University, Jinan 250014, China;College of Medical Technology, Jiangsu College of Nursing, Huaian 223300, China;Department of Clinical Laboratory, the General Hospital of the PLA Rocket Force, Beijing 100032, China;Department of Clinical Laboratory, Shanghai Chang Zheng Hospital, Shanghai 200003, China;Department of Rheumatology, Zhejiang Provincial People's Hospital, Hangzhou 310014, China)

机构地区：[1]山东第一医科大学第一附属医院检验科,济南250014 [2]江苏护理职业学院医学技术学院,淮安223002 [3]中国人民解放军火箭军总医院检验科,北京100032 [4]上海长征医院检验科,200003 [5]5浙江省人民医院风湿科,杭州310014

出　　处：《中华风湿病学杂志》2019年第8期524-529,共6页Chinese Journal of Rheumatology

基　　金：国家自然科学基金青年项目(81600110);浙江省医药卫生科技计划项目(2016KYA010);浙江省人民医院优秀青年人员科研启动基金(ZRY2016B008).

摘　　要：目的检测APS患者外周血中性粒细胞胞外诱捕网(NETs)水平,并初步探讨其形成机制。方法收集27例APS患者和30名健康对照者血浆,PicoGreen核酸定量分析试剂盒检测血浆中循环游离DNA(cf-DNA)的含量,ELISA法分析血浆中瓜氨酸化组蛋白3(CitH3)浓度,并分析cf-DNA/NETs含量与APS患者血栓事件的关联。密度梯度离心法分离健康对照组中性粒细胞,体外用抗β2糖蛋白I(β2-GPI)/β2GPI复合物(100 μg/ml)刺激4 h,测定培养上清中cf-DNA/NETs的含量,进一步采用Toll-样受体4(TLR4)抑制剂-TAK242(5 μmol/L),观察是否能够干预抗β2GPI/β2GPI复合物对细胞的刺激效应。组间差异采用方差分析或秩和检验,两两比较采用Sidak或Dunnett′s,变量间的相关性采用Spearman相关分析。结果与健康对照组相比,APS患者血浆cf-DNA/NETs浓度和CitH3含量均显著升高[175.7(70.6,205.7)ng/ml与29.8(7.6,115.7)ng/ml,Z=-3.654,P<0.05;19.5(7.8,26.4)ng/ml与3.3(0.84,10.3)ng/ml,Z=-3.932,P<0.05],且CitH3含量与cf-DNA/NETs浓度两者具有相关性(r=0.447,P=0.019);在APS患者组,动脉血栓患者与静脉血栓患者的血浆cf-DNA/NETs差异无统计学意义[177.1(67.8,297.2)ng/ml与184.7(82.4,233.9)ng/ml,Z=-0.301,P=0.786],而新近发生血栓事件的患者(时间≤1个月)血浆cf-DNA/NETs含量高于既往有动静脉血栓发生的患者[192.1(83.6,328.8)ng/ml与90.0(42.8,184.7)ng/ml,Z=-2.006,P=0.046]。体外实验中,抗β2GPI/β2GPI复合物(100 μg/ml)能够刺激中性粒细胞释放cf-DNA/NETs,与对照组相比差异有统计学意义(t=10.39,P<0.05),而TAK242明显抑制抗β2GPI/β2GPI复合物对细胞的刺激效应(t=4.22,P<0.05)。结论APS患者外周血cf-DNA/NETs水平显著增加,其可能在APS血栓形成中发挥重要作用;抗β2GPI/β2GPI复合物通过TLR4诱导cf-DNA/NETs形成,参与APS病理过程。Objective To detect the levels of neutrophil extracellular traps (NETs) in patients with antiphospholipid syndrome (APS) and to preliminarily explore its formation mechanism. Methods Plasma samples from 27 APS patients and 30 healthy controls were collected. The circulating free DNA (cf-DNA) in plasma was detected by the PicoGreen nucleic acid quantitative assay kit, and the concentration of citrulline histone 3 (CitH3) was analyzed by enzyme-linked immuno sorbent assay (ELISA). The association of cf-DNA/NETs with thrombotic events in APS patients was further analyzed. The neutrophils in healthy controls were separated by density gradient centrifugation and stimulated with anti-β2GPI/β2GPI complex (100 μg/mL) for 4 h, and the cf-DNA/NETs in the culture supernatant was determined. TLR-4 inhibitor-TAK242 (5 μmol/L) was further used to observe whether the stimulation of the anti-β2GPI/β2GPI complex on cells could be intervened. The differences between groups were analyzed by analysis of variance (ANOVA) or rank sum test, Sidak or Dunnett′s test were used to compare the mean of multiple samples and the correlation between variables was analyzed by Spearman's correlation test. Results The concentration of cf-DNA/NETs and CitH3 were significantly increased in plasma of APS patients compared with that in healthy controls [175.7(70.6, 205.7) ng/ml vs 29.8(7.6, 115.7) ng/ml, Z=-3.654, P<0.05;19.5(7.8, 26.4) ng/ml vs 3.3(0.84, 10.3) ng/ml, Z=-3.932, P<0.05], and there was a significant positive correlation between the cf-DNA/NETs and CitH3 (r=0.447, P=0.019). In the APS group, there was no significant difference in cf-DNA/NETs between patients with arterial thrombosis and those with venous thrombosis [177.1(67.8, 297.2) ng/ml vs 184.7(82.4, 233.9) ng/ml, Z=-0.301, P=0.786], whereas cf-DNA/NETs in the patients who experienced a new thrombotic event in 1 month was significantly higher than those with a history of thrombosis [192.1(83.6, 328.8) ng/ml vs 90.0(42.8, 184.7) ng/ml, Z=-2.006, P=0.046]. In vitro, anti-β2GP

关 键 词：抗磷脂综合征 中性粒细胞胞外诱捕网 Toll-样受体4 抗β2糖蛋白I/β2GPI复合物 血栓 

分 类 号：R593.2[医药卫生—内科学]