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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。
Objective To investigate the formation of neutrophil extracellular traps (NETs) in the peripheral blood of patients with bacterial sepsis and its changes with the progression of the disease. Methods Sixty patients with bacterial sepsis were collected. Among them, 10 were pathogenic bacteria Staphylococcus aureus, 10 were Escherichia coli, 10 were Pseudomonas aeruginosa and 30 were Klebsiella pneumoniae Type 15 cases, mucinous type 15 cases); another 30 cases of healthy people as the control group. Peripheral blood samples of patients with bacterial sepsis were collected on day 1 and day 7 respectively. Neutrophils were isolated by density gradient sedimentation. Sytox Green was used to confirm the presence of NETs. Immunofluorescent staining and nuclear stain Histone H3 and DNA were co-located in NETs, Image-Pro Plus 6.0 Microsoft system for quantitative detection of NETs, NETs in peripheral blood of patients with sepsis and healthy people and sepsis patients treated with antibiotics 7d NETs Changes in the situation. Results (1) The presence of NETs was detected in the peripheral blood of 60 patients with bacterial sepsis and it was demonstrated that histone H3 and DNA co-localized in NETs; (2) The content of NETs in the peripheral blood of septic patients (42.358 (T = 8.33, P <0.05); (3) The content of NETs in the patients with dissipating phase after antibiotic treatment for 7d (19.793% ± 1.917% vs 0.262% ± 0.041%,% ± 1.967% (51.191% +/- 5.550%) (t = 7.64, P <0.05). (4) In patients with bacterial sepsis caused by mucus-type Klebsiella pneumoniae (hvKP) (53.865% ± 1.385%) in peripheral blood was significantly higher than that of common Klebsiella pneumoniae (27.628% ± 1.425%) (t = 7.82, P <0.05). Conclusions NETs are formed in the peripheral blood of patients with bacterial sepsis and the amount of NETs is also reduced as the disease progresses; NETs can be induced by both Klebsiella pneumoniae and Klebsiella pneumoniae , But the patterns that form NETs are different. Klebsiella pneumoniae is more susceptible to neutrophil killing and Klebsiella pneumoniae may escape the capture of NETs by some mechanism. NETs plays an important bactericidal effect in patients with bacterial sepsis.