【作者】 焦嘉；

【导师】 周新民；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 第一部分血清降钙素原应用于心脏术后呼吸机相关性肺炎诊断及预后的临床研究目的：观察心脏术后早发型呼吸机相关性肺炎(Ventilator-associated pneumonia, VAP)患者血清降钙素原(Procalcitonin, PCT)、胰石蛋白(Pancreatic stone protein, PSP)、白介素6(Interleukin-6, IL-6)、C反应蛋白(C-reactive protein, CRP)的动力学改变,评价这些指标在VAP中的诊断及预后价值,为临床上准确诊断及评估VAP患者提供参考。方法2012年2月至2012年12月期间在中南大学湘雅二医院心胸外科重症加强护理病房(Intensive care unit, ICU)接受机械通气大于48小时的心脏术后成年患者51例,根据机械通气4天内是否诊断为VAP分为VAP组(n=33)及非VAP组(n=18),对VAP组患者随访28天,根据患者是否死亡进一步分为存活组26例和死亡组7例。所有患者术前、VAP组患者在临床诊断VAP当天(非VAP组患者在拔管当天,定义为第一天)之后的1周内隔日抽取静脉血4次进行血清PCT、PSP、IL-6及CRP测定。其中PCT、IL-6采用电化学发光法检测,CRP采用乳胶免疫比浊法检测,PSP采用酶联免疫吸附法(Enzyme-linked immuno sorbent assay, ELISA)检测。记录各组患者的一般、围手术期情况及术后非感染性并发症等资料,并统计所有患者第一天、第七天序贯器官衰竭估计(Sequential organ failure assessment, SOFA)评分和下呼吸道分泌物病原学培养阳性结果。两组间比较采用Student t检验、Mann-Whitney检验或卡方检验。采用受试者工作特征曲线(Receiver operating characteristic curve, ROC曲线)评价各指标对VAP诊断及预后的检验效能。Spearman秩相关分析方法计算两变量之间的关系。结果VAP组患者机械通气时间、ICU住院时间、第一天及第七天的SOFA评分显著高于非VAP组,死亡组患者的第一天及第七天的SOFA计分也明显高于存活组,差异均具有统计字意义。其他围手术期情况和术后非感染性并发症等在各组患者间无统计学差异。所有患者术后血清PCT、IL-6、CRP及PSP均较术前显著升高。心脏术后发生VAP的患者第一天血清PCT显著高于非VAP患者,诊断VAP的ROC曲线下面积为0.886(P值=0.00),第一天血清PCT≥5.0ng/ml诊断VAP敏感性及特异性可分别达到93.9%及83.3%,而血清IL-6、CRP及PSP对VAP均无诊断价值；在发生VAP的患者中,第五天及第七天PCT水平、第七天CRP及PSP水平、第一天及第七天SOFA评分对患者死亡均有一定预测价值,其中以第七天血清PCT预测价值最佳,ROC曲线下面积为0.869(P值=0.01),以6.4ng/ml为截断值,PCT对预测VAP患者死亡敏感性为80.0%,特异性可达96.2%。第一天及第七天SOFA评分预测VAP死亡的ROC曲线下分别为0.805及0.865(P值<0.05),第一天SOFA评分≥14分和第七天SOFA评分≥9分的敏感性及特异性分别为57.1%、80.0%及92.3%、84.6%。第七天血清PSP和CRP预测VAP死亡的ROC曲线下面积分别为0.808和0.831(P值<0.05),第七天血清PSP≥494ng/ml及血清CRP≥87ng/ml预测VAP患者死亡的敏感性和特异性分别为80.0%、80.0%和80.8%、73.1%。血清PCT水平与SOFA评分呈显著的直线正相关(第一天及第七天血清PCT和SOFA评分的Pearson积差相关系数(Pearson’s r,r)分别为0.54及0.66,P值<0.01)。33例确诊VAP患者下呼吸道分泌物培养分离出革兰阴性杆菌37株,占82%,革兰阳性球菌5株,占11%,真菌3株,占7%。结论1.经历体外循环的心脏手术对血清PCT、PSP、IL-6及CRP水平均产生影响,术后并发症能够使血清PCT、PSP、IL-6及CRP显著上升；2.PCT对诊断心脏术后早发型VAP有重要参考价值,而IL-6、CRP及PSP对VAP均无诊断价值；3.连续监测血清PCT、PSP及CRP动力学变化对有助于预测VAP患者的预后,在病程的不同时期内,SOFA评分及血清PCT、CRP及PSP水平均对VAP患者的死亡均有一定预测价值,其中以血清PCT的敏感性及特异性最高,是预测VAP患者预后的良好指标；4.血清PCT与SOFA评分存在显著正相关,能够反映出患者的病情危重情况,其水平越高,病情越重；5.VAP病原学以革兰阴性杆菌为主,血清PCT、PSP、IL-6及CRP无助于革兰阳性菌及革兰阴性菌的鉴别。第二部分Th17/Treg细胞在心脏术后呼吸机相关性肺炎中表达及作用的初步研究目的研究心脏术后全身炎症反应综合征(Systemic inflammatory response syndrome, SIRS)及呼吸机相关性肺炎(Ventilator-associated pneumonia, VAP)中辅助性T细胞17(Thelper17cell, Th17细胞)和调节性T细胞(RegulatoryT cell, Treg细胞)特异性转录因子维甲酸相关孤儿核受体(Retinoid-related orphan nuclear receptor-γt, ROR-yt)和叉头翼状螺旋转录因子3(Forkhead transcription factor3, Foxp3)及相关细胞因子IL-17和IL-10、IL-6mRNA.蛋白水平的表达情况,以及各指标与血清PCT水平的关系,初步探讨Th17/Treg细胞在心脏术后SIRS及VAP早期阶段中的作用机制,为揭示Th17/Treg细胞平衡调节机制在VAP发病及治疗中提供一种理论和实验依据。方法2012年2月至2012年12月期间在中南大学湘雅二医院心胸外科重症加强护理病房(Intensive care unit, ICU) J心脏术后接受机械通气大于48小时的SIRS患者8例及VAP患者8例,另选取正常健康人8例,采用密度梯度离心法分离外周血单个核淋巴细胞(Peripheral blood mononuclear cell, PBMC),采用RNA提取试剂盒提取PBMC中总RNA,分光光度计测定总RNA浓度,琼脂糖凝胶电泳检验提取的总RNA质量。采用实时定量逆转录聚合酶链反应技术(Reverse transcription polymerase chain reaction, RT-PCR)测定PBMC中Th17细胞和Treg细胞特异性转录因子ROR-yt和Foxp3及相关细胞因子IL-17和IL-10、IL-6mRNA的表达情况。测定血清IL-17、IL-10、IL-6及PCT水平,其中IL-17、IL-10采用酶联免疫吸附法(Enzyme-linked immuno sorbent assay, ELISA)检测,PCT、IL-6采用电化学发光法检测。mRNA的计算采用-△CT相对定量法。两组间比较采用Student t检验或Mann-Whitney检验。Spearman秩相关分析方法计算各检测指标与血清PCT之间的关系。结果抽提的PBMC中总RNAOD26onm/OD280nm比值均在1.8-2.0之间,用1%的琼脂糖凝胶电泳检测显示清晰的三条分别为28S、18S、5s的条带,其中18s与28s RNA荧光强度比值约为1：2。所有实时定量PCR熔解曲线图上均只有一个峰值。VAP组、SIRS组患者的IL-17、IL-10、IL-6、Foxp3及ROR-yt的mRNA水平均较正常对照组升高,差异有统计学意义,VAP组患者的IL-10、Foxp3的mRNA水平显著高于SIRS组(P值<0.05),而IL-17、IL-6及ROR-yt的mRNA水平在VAP组及SIRS组间无统计学差异。VAP组、SIRS组患者的血清IL-17、IL-10及IL-6水平均显著高于正常对照组,差异有统计学意义。VAP组患者血清IL-10水平也显著高于SIRS组,但血清IL-17及IL-6水平在SIRS组及VAP组间无统计学差异。Foxp3、血清IL-10水平与血清PCT水平存在直线正相关,其Pearson积差相关系数(Pearson’s r, r)分别为0.53及0.64(P值<0.05)。结论1.Thl7细胞参与心脏术后SIRS的发生,在VAP发病早期的作用仍需进一步探讨；2.Treg细胞在心脏术后活化并对抗SIRS诱发的炎症反应,Treg细胞介导的抗炎反应在VAP诊断初期已经发生,并在VAP患者体内紊乱的免疫调节功能中发挥着一定作用；3.血清PCT与Treg细胞特异性转录因子ROR-yt及血清IL-10水平存在正相关,能够在一定程度上反映患者体内的抗炎反应活化程度。更多还原

【Abstract】 Part Ⅰ Assessment of the accuracy of serum procalcitonin for the diagnosis and prognosis of ventilator-associated pneumonia after cardiac surgeryObjectiveIn order to provide reference to accurately diagnose ventilator-associated pneumonia(VAP) after cardiac surgery in clinics, we investigated the kinetics alteration of serum Procalcitonin (PCT)、 Pancreatic stone protein (PSP)、Interleukin-6(IL-6) and C-reactive protein (CRP) from early onset VAP patients after cardiac surgery with cardiopulmonary bypass(CPB) and assessed these biomarkers’value for diagnosis and prognosis of VAP after cardiac surgery.MethodsThis study included51consecutive patients aged≥18years who accepted48hours or more of mechanical ventilation at our intensive care unit(ICU) of cardiac surgery (Second Xiang-Ya Hospital,Changsha,China) between February2012and December2012.All of them undergone elective cardiac surgery with the use of CPB.33patients were diagnosed with early onset VAP (mechanical ventilation time less than4days) and the remaining18patients formed a non-VAP group. Follow-up were for28days or until death. In VAP group, patients who died within28days after VAP onset were classified as non-survivors (7patients),others (26patients) were classified as survivors. Blood samples for biologic measurements were drawn before cardiac surgery(baseline). In VAP group,Dayl was defined as the day when the VAP diagnosis was confirmed while Day1was defined as the day when the patients removed the tracheal intubation in the non-VAP group. Serum PCT、PSP、IL-6and CRP level were measured every two days(Day1、Day3、Day5、Day7) in VAP group and non-VAP group.Serum PCT and IL-6were measured by electrochemiluminescence immunoassay. Serum CRP was measured by immune turbidimetry while serum PSP was measured by Enzyme-linked immuno sorbent assay (ELISA).Date collected included preoperative and postoperative characteristics and non-infectious complications. Sequential Organ Failure Assessment (SOFA) were evalued in all the patients on Day1and Day7. Date of positive lower respiratory tract secretions from VAP patients was also collected. Comparsion between two groups were performed using the Student t test、the Mann-Whitney test or the Fisher exact method,when appropriate.Receiver operating characteristic (ROC) curves was used to determine the best threshold values for sensitivity and specificit.Correlations were calculated by Spearman rank correlation. ResultsDuration of mechanical ventilation、Length of stay in the ICU and SOFA on Day1and Day7were significantly higher in VAP group than non-VAP group. Non-survivors group’s SOFA on Dayl and Day7were also significantly higher than those of survivors group.There were no differences in pre-operation characteristics and non-infectious complications between the non-VAP group and VAP group. Serum PCT、 PSP、IL-6and CRP level from all the patients were significantly high compared with their pre-operation baseline level. On Day1,Serum PCT level in patients who were diagnosed with VAP after cardiac surgery were markly higher than those of the non-VAP group. On Dayl, the best PCT cutoff value for VAP diagnosis was5.0ng/ml,with a sensitivity of93.9%and a specificity of83.3%(area under the curve:0.886; P<0.01). None of serum PSP、IL-6、CRP had significant predictive value for VAP diagnosis. Among the patients who were diagnosed with VAP,serum PCT level on Day5and Day7、serum PSP and CRP level on Day7, SOFA on Dayl and Day7all had some predictive value in detecting non-survivors. On Day7,the area under the ROC curve that used PCT to detect non-survivors was0.869(p=0.01),which provided the best predictive value. The best PCT cutoff value for VAP prognosis was6.4ng/ml, with a sensitivity of80.0%and a specificity of96.2%.Used SOFA to detect non-survivors was considerable:the area under the ROC curve of SOFA on Dayl and Day7were0.805and0.865, respectively. For detection of non-survivors by SOFA, a value of14on Day1showed57.1%sensitivity,92.3%specificity and a value of9on Day7showed80.0%sensitivity,84.6%specificity.The area under the ROC curve that used serum PSP and CRP on Day7to detect non-survivors were0.831and0.808. Serum PSP≥494ng/ml and CRP≥87ng/ml on Day7for VAP prognosis provided80.0%.80.0%sensitivity and80.8%、73.1%sensitivity, respectively. There was a postive linear correlation between serum PCT level and SOFA (the Pearson’s r between PCT level and SOFA on Day1and Day7were0.54and0.66respectively, P<0.01).37strains gram-negative bacilli were separated from the lower respiratory tract secretions from33patients with VAP,with a proportion of82%.5strains gram-postive bacteria made up11%and3strans fungus accounted for7%.Conclutions1. Cardiac surgery with CPB has an influence on serum PCT、PSP IL-6and CRP level and they are markly higher in patipents with postoperation complications.2. PCT is a useful marker for diagnosing VAP after cardiac surgery and none of PSP、IL-6、CRP have significant predictive value for VAP diagnosis.3. Continuous monitoring kinetics alteration of serum PCT、PSP、 IL-6and CRP in VAP patipents may contribute to predict the prognosis of VAP. At different phases of VAP, PCT、SOFA、PSP and CRP all have some predictive value in detecting non-survivors.PCT which provides the best sensitivity and specificity may be a good marker for VAP prognosis.4. Majority of pathogenic bacteria in VAP are Gram-negative bacilli. Serum PCT、PSP、IL-6and CRP does not differ between Gram-negative bacilli and Gram-negative bacilli. Part II Research on the expression and effects of Th17/Regulatory T cell in ventilator-associated pneumonia after cardiac surgeryObjectiveTo investigate the imbalance between T helper17cell(Th17cell) and CD4+CD25+Foxp3+regulatory T (Treg) cell, the expression of Thl7/Treg cell key transcription factors Retinoid-related orphan nuclear receptor-yt (ROR-yt)、Forkhead transcription factor3(Foxp3) and related cytokines IL-17、IL-10and IL-6mRNA and protein level、the relationship with serum procalcitonin (PCT) and the possible mechanism during systemic inflammatory response syndrome (SIRS) and early stage of ventilator-associated pneumonia (VAP) after cardiac surgery.Try to discover the relationship between the imbalance of Th17/Treg cell and the ventilator-associated pneumonia and afford the theoretical and experimental proof for the onset and treatment of VAP after cardiac surgery.MethodsThis study included8patients who were diagnosed with VAP and8patients with SIRS who accepted48hours or more of mechanical ventilation but not developed VAP at our intensive care unit(ICU) of cardiac surgery (Second Xiang-Ya Hospital,Changsha,China) between February2012and December2012.Meanwhile,8healthy volunteers formed a normal control group. Peripheral blood mononuclear cell (PBMC) were isolated by Ficoll-Hyaque density gradient centrifugation.Total RNA were extracted using a RNA extraction kit. Total RNA concentrations were determined by spectrophotometer. Total RNA quality were assessed by agarose gel electrophoresis. The expression of Th17/Treg cell key transcription factors ROR-yt. Foxp3and the related cytokines IL-17、IL-10and IL-6mRNA were measured by real time quantitative Reverse transcription polymerase chain reaction (RT-PCR). Serum IL-17、IL-10were measured by Enzyme-linked immunosorbent assay (ELISA) while Serum IL-6、PCT were measured by electrochemiluminescence immunoassay. mRNA calculations were used-△CT relative quantitative method.Comparsion between the two groups were performed using the Student t test or the Mann-Whitney test,when appropriate. Correlations between these index and serum PCT were calculated by Spearman rank correlation.ResultsThe ratio of OD260nm/OD280nm of the total RNA were all between1.8and2.0.28s、18s and5s pattern appeared upon agarose gel electrophoresis.The RNA fluorescence intensity ratio of18s to28s was almost1:2. There were only a peak in all the realtime quantitative PCR melted curves.IL-17、IL-10、IL-6. Foxp3and ROR-γt mRNA in SIRS group and VAP group were higher than those in the normal control group.The difference was significant. IL-10and Foxp3mRNA in VAP group were also significantly higher than those in SIRS group. IL-17、I1-6and ROR-yt mRNA did not differ between SIRS group and VAP group.Serum IL-17、IL-10and IL-6level in SIRS group and VAP group were higher than those in the normal control group.The difference was significant. Serum IL-10level in VAP group were also significantly higher than SIRS group.No differences in the level of serum IL-17and I1-6were found between SIRS group and VAP group. There was a postive linear correlation between serum PCT level and Foxp3、serum IL-10(the Pearson’s r were0.53and0.64respectively, P<0.05)Conclution1.Th17cell involves in the development of SIRS after cardiac surgery.The role of Th17cell in the early stage of ventilator-associated pneumonia needs further discussed.2. Treg cell is activated after cardiac surgery and fights against SIRS-induced inflammatory reaction. Treg cell-mediated anti-inflammation response has already occurred during early stage of ventilator-associated pneumonia and play a role in immunity disorder of VAP patients.3. Serum PCT level has a postive linear correlation with Treg cell key transcription factors Foxp3and serum IL-10level.PCT is parallel with the degree of anti-inflammation activation response to a certain extent.更多还原