目的 探讨一氧化氮供体对哮喘儿童来源的中性粒细胞胞外诱捕网(NETs)的诱导作用,寻找新的可能用于儿童哮喘的监测指标和治疗靶点。方法 选取2017年9月-2017年11月就诊于上海市第五人民医院儿科的38例支气管哮喘患儿为研究对象,利用Percoll密度梯度离心法从哮喘儿童的外周血中分离出中性粒细胞,加入一氧化氮供体SNP进行体外孵育,荧光染色法检测细胞上清中的双链DNA(NETs的主要组分)做定量分析,同时制备细胞爬片并利用免疫荧光的方法观察NETs的主要结构,此外还利用一氧化氮合酶(NOS)的抑制剂对SNP的诱导进行干预实验。结果 成功从哮喘儿童外周静脉血中分离出中性粒细胞,回收率达95%以上,细胞活性大于80%;PMA、SNP组在诱导3 h后明显可见释放到胞外的丝网状NETs结构,而PBS、SNP+L-NAME、SNP+SMT组在诱导3 h后均未见明显的NETs结构;干预组(PMA 160 nmol/L、LPS 100 ng/ml、SNP 400 μmol/L)的NETs相对生成量均大于阴性对照PBS组(P<0.05);SNP+NOS阻滞剂组(SMT 10mmol/L、L-NAME 500 μmol/L、L-NMMA 1 mmol/L)的NETs相对生成量均小于单纯SNP组(P<0.05);NOS抑制剂能够减少但不能完全阻滞NETs的生成(P<0.05);SNP诱导的第1 h、2 h、3 h的NETs相对生成量呈持续上升趋势,而PBS组在第3 h生成量下降(P<0.05);随着L-NAME干预时间的延长,SNP诱导的NETs相对生成量持续减少(P<0.05)。结论 SNP能够诱导哮喘儿童NETs形成,同时NOS的抑制剂能够减少NETs的产生,为儿童哮喘的监测和治疗提供了新的思路。

Objective To investigate the inductive effect of nitric oxide donor on asthmatic children′s neutrophil extracellular traps (NETs),in order to find new monitoring indicators and therapeutic targets that may be used in childhood asthma. Methods Totally 38 children with bronchial asthma in the Pediatric Department of the Fifth People′s Hospital of Shanghai were enrolled in this study from September to November in 2017. Percoll density gradient centrifugation was used to isolate neutrophils from the peripheral blood of asthmatic children, and nitric oxide donor (SNP) was added for in vitro incubation.Fluorescent staining was used to detect the double-stranded DNA (the main component of NETs) in the cell supernatant for quantitative analysis.Meantime, the cell slides were prepared to observe the main structures of NETs by fluorescence microscopy.In addition, inhibitors of nitric oxide synthase (NOS) were used to interfere the induction of SNP. Results The neutrophils were successfully isolated from the peripheral blood of asthmatic children, and the recovery rate was over 95%, the cell viability was over 80%.The extracellular mesh-like NETs structures were obviously observed in 3 hours after induction in PMA and SNP group, but NETs structure was not observed in PBS, SNP + L-NAME and SNP + SMT groups.The relative amounts of NETs in the intervention group (PMA 160 nmol/L, LPS 100 ng/ml and SNP 400 μmol/L) were significantly higher than those in the negative control PBS group (P＜0.05).The relative amounts of NETs in SNP + NOS blockers group (SMT 10 mmol/L, L-NAME 500 mol/L and L-NMMA 1 mmol/L) were less than simple SNP group (P＜0.05).NOS inhibitor could reduce but could not completely block the generation of NETs (P＜0.05).The relative generation of NETs at 1 h, 2 h, 3 h induced by SNP kept increasing while the formation decreased at 3 h with PBS(P＜0.05).With the prolongation of L-NAME, the relative amount of NETs induced by SNP decreased continuously(P＜0.05). Conclusion SNP can induce the formation of NETs in asthmatic children, and inhibitors of NOS can reduce the generation of NETs, which provides a new idea for the monitoring and treatment of childhood asthma.