中性粒细胞胞外陷阱在细菌性脓毒症中的作用

Role of neutrophil extracellular traps in treating bacterial sepsis

目的探究中性粒细胞胞外陷阱(neutrophil extracellular traps,NETs)在细菌性脓毒症患者外周血中的形成及其随着疾病进程的变化情况。方法收集细菌性脓毒症患者60例,其中致病菌为金黄色葡萄球菌的患者有10例、大肠埃希菌10例、铜绿假单胞菌10例和肺炎克雷伯菌30例(普通型15例,黏液型15例);另选30例健康体检者为对照组。分别于患者入院的第1天和第7天采集细菌性脓毒症患者的外周血,密度梯度沉淀法分离纯化中性粒细胞,荧光染料Sytox Green明确NETs的存在,免疫荧光染色及核染色剂证明组蛋白H3与DNA共定位于NETs,Image-Pro Plus 6.0微软系统对NETs进行定量检测,比较脓毒症患者与健康者外周血中NETs的形成情况及脓毒症患者经过抗生素治疗7d后NETs的变化情况。结果 (1)60例细菌性脓毒症患者的外周血中检测到了NETs的存在,并且证明了组蛋白H3与DNA共定位于NETs;(2)脓毒症患者外周血中NETs的含量(42.358%±1.967%)明显高于健康体检者(0.262%±0.041%)(t=8.33,P<0.05);(3)抗生素治疗7d后,处于感染消散期患者体内NETs的含量(19.793%±1.917%)明显低于仍处在感染期的患者(51.191%±5.550%)(t=7.64,P<0.05);(4)黏液型肺炎克雷伯菌(hvKP)引起的细菌性脓毒症患者的外周血中NETs的含量(53.865%±1.385%)明显高于普通型肺炎克雷伯菌(cKP)(27.628%±1.425%)(t=7.82,P<0.05)。结论 NETs在细菌性脓毒症患者外周血中形成,且随着疾病进程的好转,NETs的量也会减少;黏液型肺炎克雷伯菌与普通型肺炎克雷伯菌均可诱发NETs的形成,但形成NETs的模式有差异。普通型肺炎克雷伯菌更易被中性粒细胞杀灭,黏液型肺炎克雷伯菌可能通过某些机制逃离NETs的捕获。NETs在细菌性脓毒症患者中起到重要杀菌作用。

Abstract： Objective To investigate the formation of neutrophil extracellular traps （NETs） in the peripheral blood of patients with bacterial sepsis, and its changes along with the development of the disease. Methods 60 cases of bacterial sepsis from a prospective study in May and accepted by the ACCP/SCCM conference were collect- ed, including 10 patients infected by Staphylococcus aureus, 10 by Escherichia coli, 10 by Pseudomonas aeruginosa, and 30 by K. pneumonia, with 30 healthy individuals as the controls. Blood samples were ob- tained from the patients on Day 1 and Day 7 and stained with Sytox Green to verify the existence of NETs. The co-localization of histone H3 and DNA at NETs was confirmed with immunofluorescence and nucleus staining. Quantitative determination of NETs was done with Microsoft Image-Pro Plus 6.0 System. The levels of NETs of the patients were compared with those of the healthy controls, and with those before treat- ment with antibiotics for 7 days. Results （1） NETs were detected in the 60 blood samples from the pa- tients, and the co-localization of Histone H3 and DNA NETs was confirmed. （2） The baseline levels of NETs of patients （n= 30） were higher than those of healthy controls （42. 358±1. 967） vs O. 262% ±0. 041X, P〈0.05） . （3） Patients who still suffered from the infection had significantly higher NETs levels （51.191±5. 550%） than those who were recovering （19. 793%±1. 917%） （P〈0.05） after being trea- ted with antibiotics for 7 days. （4） The NETs levels of patients infected by hvKP were higher than those in- fected by cKP （53. 865%±1. 385% vs 27. 628%--±1. 425%, P〈0.05） . Conclusion NETs were formed in peripheral blood of bacterial sepsis patients, the levels of which showed a decreasing trend along with the recovery of the disease. Both hvKP and cKP were able to induce the formation of NETs, but in different ways. cKP tend to be inactivated by neutrophils, yet hvKP might be able to escape from NETs in some way. NETs could be used as a good disinfectant by bacterial sepsis patients.