摘要
目的探讨脓毒症肝损伤患者外周血中性粒细胞(PMN)功能变化及其对预后评价的指导作用。方法采用前瞻性观察性研究方法,选择2019年3月至8月南京医科大学附属无锡人民医院重症医学科(ICU)收治的符合脓毒症3.0诊断标准的患者作为研究对象,根据肝损伤诊断标准将患者分为脓毒症非肝损伤组和脓毒症肝损伤组;选择同期住院的非脓毒症患者作为非脓毒症组;选择本院体检中心健康体检者作为健康对照组。记录患者入ICU时及健康体检者体检当天的性别、年龄、白细胞计数(WBC)、PMN、降钙素原(PCT),计算患者急性生理学与慢性健康状况评分Ⅱ(APACHEⅡ)和序贯器官衰竭评分(SOFA);记录患者28 d病死率;取外周静脉血,使用PicoGreen荧光染料定量检测血浆中性粒细胞胞外诱捕网(NETs)水平,以游离DNA(cf-DNA/NETs)水平表示;采用细胞免疫荧光染色进行NETs定性检测。提取健康对照组、脓毒症非肝损伤组、脓毒症肝损伤组PMN并进行体外培养,使用PicoGreen荧光染料定量检测细胞上清cf-DNA/NETs水平。根据脓毒症肝损伤患者28 d临床转归分组,绘制受试者工作特征曲线(ROC),并计算ROC曲线下面积(AUC),分析外周血NETs水平对脓毒症肝损伤患者预后的预测价值。结果最终纳入脓毒症非肝损伤患者21例,脓毒症肝损伤患者15例,非脓毒症患者20例,健康体检者20例。4组间性别、年龄差异均无统计学意义,说明各组一般资料均衡,具有可比性。脓毒症非肝损伤与肝损伤组外周血cf-DNA/NETs水平及WBC、PMN均明显高于健康对照组和非脓毒症组,PCT、APACHEⅡ评分、SOFA评分、28 d病死率均明显高于非脓毒症组,且脓毒症肝损伤组外周血cf-DNA/NETs水平及PCT、28 d病死率均明显高于脓毒症非肝损伤组〔cf-DNA/NETs(μg/L):481.60±275.86比169.76±57.05,PCT(μg/L):11.29(1.79,67.10)比1.11(0.19,4.09),28 d病死率:73.3%(11/15)比38.1%(8/21),均P<0.05〕。体外培养PMN结果显示,正常培养时健康对照组无NETs产生,脓毒症非肝损伤与肝损伤组均有少量NETs产生;加入PMN刺激剂佛波醇-12-肉豆蔻酯-13-乙酸(PMA)刺激后,3组PMN均较正常培养时产生更多的NETs。定量分析显示,脓毒症肝损伤组细胞上清cf-DNA/NETs水平明显高于脓毒症非肝损伤组(μg/L:1872.29±258.44比1313.55±147.45,P<0.01)。15例脓毒症肝损伤患者28 d存活4例(占26.7%),死亡11例(占73.3%);死亡组患者入院时外周血cf-DNA/NETs水平明显高于存活组(μg/L:582.36±160.05比241.17±96.14,P<0.05)。ROC曲线分析显示,外周血NETs水平预测脓毒症肝损伤患者28 d死亡的AUC为0.932〔95%可信区间(95%CI)为0.787~1.000〕;当最佳截断值为266.81μg/L时,敏感度为90.9%,特异度为75.0%,约登指数为0.659。结论脓毒症肝损伤患者NETs的功能发生了改变;外周血NETs水平对脓毒症肝损伤患者的预后有一定预测价值。
Objective To explore the changes in polymorphonuclear neutrophils(PMN)function in peripheral blood of patients with sepsis and liver injury and its prognostic value.Methods A prospective observational study was conducted.The patients who met the criteria of Sepsis-3 admitted to intensive care unit(ICU)of Wuxi People's Hospital Affiliated to Nanjing Medical University from March to August in 2019 were enrolled as the research objects,and the patients were divided into sepsis without liver injury group and sepsis with liver injury group;non-sepsis patients who were hospitalized at the same time were enrolled as non-sepsis group;and the healthy people in the physical examination center were enrolled as healthy control group.The gender,age,white blood cell(WBC),PMN and procalcitonin(PCT)were recorded when the patients were admitted to ICU as well as the people on the day of physical examination.The acute physiology and chronic health evaluationⅡ(APACHEⅡ)and sequential organ failure assessment(SOFA)scores were calculated.The 28-day mortality was recorded.The quantitative level of neutrophil extracellular traps(NETs)which reflected by circulating free DNA(cf-DNA/NETs)in peripheral plasma was determined by PicoGreen fluorescence quantitative detection;the qualitative level of NETs was detected by immunofluorescence staining.PMN was extracted from the healthy control group,sepsis without liver injury group and sepsis with liver injury group and cultured in vitro,the quantitative level of cf-DNA/NETs in cell supernatant was determined by PicoGreen fluorescence quantitative detection.The patients were divided into two groups according to 28-day outcome of sepsis patients with liver injury.Receiver operating characteristic(ROC)curve was plotted,and the area under ROC curve(AUC)was calculated to analyze the prognostic value of NETs in sepsis patients with liver injury.Results Finally,21 sepsis patients without liver injury,15 sepsis patients with liver injury,20 with non-sepsis and 20 with healthy examination were enrolled.There was no significant difference in gender or age among the four groups,indicating that the patients in each group were comparable.The levels of cf-DNA/NETs in peripheral blood,WBC and PMN of the sepsis with and without liver injury groups were significantly higher than those of the healthy control group and non-sepsis group,PCT,APACHE Ⅱ score,SOFA score and 28-day mortality were significantly higher than those of the non-sepsis group,and the levels of cf-DNA/NETs in peripheral blood,PCT and 28-day mortality of the sepsis with liver injury group were significantly higher than those of the sepsis without liver injury group[cf-DNA/NETs(μg/L):481.60±275.86 vs.169.76±57.05,PCT(μg/L):11.29(1.79,67.10)vs.1.11(0.19,4.09),28-day mortality:73.3%(11/15)vs.38.1%(8/21),all P<0.05].The results of PMN in vitro showed that there was no NETs in normal culture of healthy control group,and a small amount of NETs was detected in sepsis with and without liver injury groups.After stimulation of PMN stimulator phorbol-12-myristate-13-acetic acid(PMA),more NETs were produced in neutrophils of three groups compared with normal culture.Quantitative analysis showed that the level of cf-DNA/NETs in cell supernatant of the sepsis with liver injury group was significantly higher than that of the sepsis without liver injury group(μg/L:1872.29±258.44 vs.1313.55±147.45,P<0.01).In 15 sepsis patients with liver injury,4 patients survived for 28 days(26.7%)and 11 died(73.3%).The cf-DNA/NETs level of the dead group on the day of admission was significantly higher than that of the survival group(μg/L:582.36±160.05 vs.241.17±96.14,P<0.05).ROC curve analysis showed that the AUC of NETs level in peripheral blood for predicting 28-day death of sepsis patients with liver injury was 0.932[95%confidence interval(95%CI)was 0.787-1.000];when the best cut-off value was 266.81μg/L,the sensitivity was 90.9%,the specificity was 75.0%,and the approximate index was 0.659.Conclusions The function of NETs in sepsis patients with liver injury has been further changed.The level of peripheral blood NETs has a certain guiding value for the prognosis of sepsis patients with liver injury.
作者
高飞
惠姣洁
杨岚
张江茜
于璇
陆士奇
Gao Fei;Hui Jiaojie;Yang Lan;Zhang Jiangqian;Yu Xuan;Lu Shiqi(Department of Emergency,the First Affiliated Hospital of Soochow University(the First Clinical Medical College of the Medical Department of Soochow University),Suzhou 215006,Jiangsu,China;Department of Intensive Care Unit,Wuxi People's Hospital Affiliated to Nanjing Medical University,Wuxi 214000,Jiangsu,China)
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2019年第11期1324-1329,共6页
Chinese Critical Care Medicine
基金
国家自然科学基金(81401619)
江苏省医药卫生青年医学人才项目(QNRC2016178)。
