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

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-β2GPI/β2GPI complex (100 μg/ml) stimulated the release of cf-DNA/NETs from neutrophils, which was significantly increased compared with the control group (t=10.39, P<0.05), while TAK242 significantly inhibited the stimulating effects of anti-β2GPI/β2GPI complex on cells(t=4.22, P<0.05). Conclusion The level of cf-DNA/NETs in peripheral blood of APS patients is significantly increased, which may play an important role in APS thrombosis. Anti-β2GPI/β2GPI complex induces the formation of cf-DNA/NETs through TLR4 and participates in the pathological process of APS.