【作者】 金悦；

【导师】 方向明；

【作者基本信息】 浙江大学 ， 麻醉学， 2012， 博士

【摘要】 研究目的：本研究分析了重症脓毒症患者出院后2到6年的长期生活质量,分析重症脓毒症患者远期生理机能、生理职能、躯体疼痛、一般健康状况、精力、社会功能、情感职能、精神健康以及日常生活能力等各方面的改变,旨在为公共卫生政策的制定、医疗资源的配置、重大研究方向的制定和研究资金的投入提供重要的理论依据。研究方法：本研究纳入2004年1月至2008年12月,共4家大学附属医院ICU的112例重症脓毒症患者和112例年龄、性别和察尔森合并症指数相匹配的非脓毒症危重病对照者进行随访研究。2010年8月至2010年12月对患者进行随访。共66例重症脓毒症患者(58.9%)和80例非脓毒症危重患者(71.4%)存活,排除失访和拒绝等病例,最终共有75例纳入随访分析并完成了面对面的随访,包括42例重症脓毒症患者和33例非脓毒症对照患者。此外,本研究对126名年龄、性别与重症脓毒症患者匹配的社区人群进行了生活质量随访。采用健康调查简表(SF-36)、焦虑自评量表(SAS)、抑郁自评量表(SDS)和日常生活能力量表(ADL)对患者的生活质量、精神状态和日常生活能力等进行评估。采用Student’s t检验等方法对各项生活质量评分进行统计分析。研究结果：重症脓毒症患者的长期生活质量和非脓毒症危重病对照者相比未发现明显差异。然而,重症脓毒症患者与社区人群相比,表现为SF-36量表多个维度的临床意义和统计学意义的降低,包括：生理机能(P=0.016)、精力(P=0.037)、情感职能(P=0.043)、精神健康(P=0.038)等维度和精神健康指数(P=0.042)。重症脓毒症患者焦虑或抑郁比例与非脓毒症危重病对照者无明显差异(焦虑：9.52%vs.9.09%；抑郁：11.90%vs.9.09%,P>0.05)无明显差异。此外,重症脓毒症患者出院1年后和随访时间点的返回工作比例与非脓毒症危重病对照者无统计学差异(60.5%vs.70.0%,P=0.417；71.1%vs.76.7%,P=0.602)。同时,返回工作的患者在生理机能(P=0.019)、一般健康状况(P=0.003)、社会功能(P=0.034)等维度和躯体健康总评(P=0.001)方面明显优于未返回工作的患者。研究结论：重症脓毒症患者出院后远期生活质量在生理机能、精力、情感职能、精神健康和精神健康总评等方面较社区人群明显下降。但是,重症脓毒症患者与非脓毒症危重病对照者者的长期生活质量、焦虑、抑郁和日常生活能力等各方面无统计学差异。研究目的：动态观察Balb/c小鼠脓毒症造模后12h、24h、72h和168h四个时间点脾脏细胞的凋亡指数和血浆IL-10、TNF-α、IL-4和IL-1β等炎症因子的水平,并分析脾脏细胞凋亡指数和血浆炎症因子表达水平的相关性,从而为下一步脓毒症早期预警生物标志物的选择提供一定的理论依据。研究方法：将健康Balb/c小鼠(20-25g)随机分为脓毒症组和假手术组,采用小鼠盲肠结扎穿孔(CLP)术复制脓毒症模型,假手术组常规开腹但不作盲肠结扎、穿孔操作。分别于12h、24h、72h和168h处死小鼠、留取脾脏和血液标本,采用原位末端标记法(TUNEL)检测脾脏细胞凋亡情况,采用酶联免疫吸附试验(ELISA)检测炎症因子水平动态变化,采用Pearson相关分析法分析凋亡指数与炎症因子表达水平的相关性。研究结果：CLP模型组小鼠造模后12h、24h、72h和168h死亡率分别为10%、46%、70%和80%,符合脓毒症高死亡率的特点；并且观察到小鼠多脏器的病理改变,提示多器官功能障碍。假手术组小鼠100%存活。12h、24h、72h和168h时,脓毒症小鼠脾脏凋亡水平均明显高于假手术组(P<0.001),并且在24h时达到高峰。进一步分析各时间点炎症因子水平,发现脓毒症小鼠12h时间点血浆IL-10、TNF-α、IL-4和IL-1β等炎症因子水平均明显高于假手术组(P<0.001)。24h时,各炎症因子水平达最高峰,并且脓毒症组小鼠和假手术组小鼠的血浆TNF-α(P=0.046)和IL-1β(P=0.035)水平存在显著性差异。对脓毒症小鼠脾脏凋亡水平和炎症因子水平进行相关性分析,发现脾脏凋亡水平与IL-1β动态变化水平存在相关性(r=0.976,P=0.024)。研究结论：小鼠脓毒症造模后,早期即出现脾脏细胞凋亡和血浆炎症因子水平明显升高。同时,脓毒症小鼠脾脏凋亡指数与炎症因子水平动态呈相同的变化趋势,脓毒症小鼠脾脏凋亡水平与血浆IL-1β水平存在相关性。第三部分循环核小体在脓毒症患者中的早期预警作用研究目的：基于目前国内外研究进展和本论文第二部分的研究发现——淋巴细胞凋亡在脓毒症的病理生理改变中发挥着重要作用,虽然Hotchkiss等观察了脓毒症病人死亡后脾脏淋巴细胞凋亡程度,但是,目前脓毒症病人脾脏淋巴细胞凋亡的检测存在其局限性。因此,本部分研究选取与凋亡密切相关的核小体,探讨循环中核小体对于脓毒症患者诊断和预后的相关性,明确循环核小体在脓毒症中的早期预警作用。研究方法：将2009年1月至9月入住2家大学附属医院外科ICU长于48小时的74名患者纳入为研究组1,同期入住另外2家浙江大学附属医院外科ICU长于48小时的91名外科术后患者为研究组2。排除化疗、AIDS、激素治疗和器官移植等患者。研究组1和研究组2所有纳入病人在入住ICU24小时内均抽取全血检测血清中核小体水平。同时,研究组1在第3天、第5天、第7天和观察终点(出ICU当天/死亡)时抽取全血。所有患者在入住ICU24小时内进行APACHE Ⅱ (Acute Physiology and Chronic Health Evaluation Ⅱ)评分,并且每天进行SOFA (Sequential Organ Failure Assessment)评分,并且以细胞因子水平作为脓毒症炎症反应严重程度指标。检测研究组1中患者血清循环核小体水平并分析其与脓毒症发生的相关性,并且在研究组2中进行验证。研究结果：两组研究组中,脓毒症组患者入住ICU时血清循环核小体水平较非脓毒症对照组均明显升高,研究组1中循环核小体鉴别脓毒症与非脓毒症的受试者工作特征曲线(ROC)下面积为0.70(95%可信区间为0.59-0.83),研究组2循环核小体鉴别脓毒症与非脓毒症的ROC曲线下面积为0.66(95%可信区间为0.51-0.81),将两组研究组所有患者合并后分析ROC曲线下面积为0.67(95%可信区间为0.55-0.79)。多元Logistic回归分析后表明循环核小体是脓毒症独立危险预测因素。同时,研究发现入住ICU24小时内的血清循环核小体水平与炎症反应严重程度和多器官功能障碍存在相关性。死亡患者入住ICU24小时内的血清循环核小体水平明显高于存活患者。研究结论：本研究显示循环核小体水平与脓毒症的发生、炎症反应严重程度、多器官功能障碍及预后存在相关性。因此,循环核小体能作为脓毒症早期预警的有效生物标志物。更多还原

【Abstract】 Part1Long-term quality of life in severe sepsis survivors:up to6years multicenter study in ChinaObjectives:The present study was undertaken to evaluate the long-term health-related quality of life (including physical functioning; role-physical; bodily pain; general health; vitality; social functioning; role-emotional and mental health) in survivors of severe sepsis up to6years. Additionally, returning to work/school was assessed.Methods:From January2004to December2008,112severe sepsis and112age-, gender-and Charlson comorbidity index-matched non-septic critically ill patients from4university hospital ICUs were enrolled. Totally66(58.9%) severe sepsis and80(71.4%) non-septic critically ill patients survived during the long-term follow-up time. Between August and December2010, a total of75patients, including42survivors of severe sepsis and33critically ill controls completed the face-to-face interview. Meanwhile,126age-and gender-matched community residents were interviewed as community control group.Results:There was no difference in the long-term HRQOL in terms of Short-Form36between severe sepsis and non-septic critically ill survivors. However, when compared with that in community controls, HRQOL in survivors of severe sepsis showed a significantly and clinically meaningful decrease, with a lower physical functioning (P=0.016), vitality (P=0.037), role-emotional (P=0.043), mental health (P=0.038) and mental component score (P=0.042). No difference was found with respect to anxiety or depression between severe sepsis and critical ill control (anxiety:9.52%vs.9.09%; depression:11.90%vs.9.09%). In addition, returning to work at1year and by the time of interview in severe sepsis were similar with that in critical illness (60.5%vs.70.0%, P=0.417,71.1%vs.76.7%, P=0.602). The patients who returned to work/school had better HRQOL in physical and mental domains than the patients who didn’t.Conclusions:The HRQOL in severe sepsis was impaired even up to6years after hospital discharge and the employment status was related to the HRQOL. However, no difference was found between severe sepsis and non-septic critically ill survivors in the long-term HRQOL, anxiety, depression, or return to work. Part2Dynamic changes of apoptosis and inflammatory factors in sepsisObjectives:Sepsis is a leading cause of death in critically ill patients, and apoptosis plays a major role in the pathophysiology of sepsis. The aim of this study was to evaluate the apoptosis of spleen tissue and the level of inflammatory factors in sepsis.Methods:Balb/c mice were randomly divided into sepsis group and sham group. In sepsis group, mice received cecal ligation and puncture (CLP) to induce sepsis. In the sham group, the cecum is exteriorized but neither ligated nor punctured. Mice of both groups were sacrificed at12,24,72,168hours after CLP. Spleens and blood were collected from every mouse. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL). The plasma levels of IL-10、 TNF-α、IL-4and IL-β were determined by enzyme-linked immunosorbent assay (ELISA).Results:At12,24,72and168hours, the mortalities were10%,46%,70%and80%, meeting the death rate requirements for sepsis models. Pathological changes were observed in the multiple organs of septic mice.. In the sham group, at168hours, the survival rate was100%. The apoptosis index (AI) of spleen cells significantly increased at12,24,72and168hours after CLP (P<0.001). The plasma level of IL-10. TNF-α、 IL-4and IL-1(3were significantly increased12hours after CLP (P<0.001). In addition, the plasma level of TNF-a (P=0.046) and IL-1β (P=0.035) were still significantly increased24hours after surgery in the septic mice than the sham group. The apoptosis index of spleen cells was associated with the plasma level of IL-1β(r=0.976, P=0.024). Conclusions:In the early stage of sepsis, the apoptosis index of spleen cells and the plasma level of IL-10、TNF-α、IL-4and IL-β were significantly increased in the septic mice than the sham controls. The variation tendency of apoptosis index was similar with the plasma level of IL-10、TNF-α、IL-4and IL-β.In addition, the variation tendency of apoptosis index was consistent with the change of IL-1β. Part3Circulating Nucleosomes as a Predictor of Sepsis and Organ Dysfunction in Critically Ⅲ PatientsObjectives:Sepsis is a leading cause of death in critically ill patients, and apoptosis plays a major role in the pathophysiology of sepsis. Elevated levels of circulating nucleosomes released by apoptotic cells have been detected in patients with severe sepsis and septic shock. The aim of this study was to evaluate the diagnostic/prognostic value of circulating nucleosomes in sepsis.Methods:Seventy-four newly admitted patients who were estimated to stay for more than48hours in intensive care unit were prospectively enrolled as Cohort1. The second independent cohort consisted of91post-surgery patients. Patients with chemotherapy, AIDS, steroids treatment, or transplants were excluded. Levels of circulating nucleosomes within24-hour of admission in both of the cohorts, and on the3rd,5th,7th day and a last time-point at ICU discharge or at imminent death in Cohort1were measured and analyzed for the capacity of predicting sepsis. Severity of inflammatory response and organ dysfunction was assessed by cytokine levels and sepsis scores.Results:The nucleosomes levels on admission in septic patients were significantly higher than those in non-septic controls in both of the cohorts. The area under the receiver-operating-characteristic curve for admission nucleosomes levels to differentiate the septic patients from non-septic patients was0.70(95%confidence interval [CI]0.59-0.83) in Cohort1and0.66(95%CI0.51-0.81) in Cohort2, and0.67(95%CI0.55-0.79) in all of the subjects. After multiple logistic regression analysis, circulating nucleosomes remained as an independent predictor for sepsis. Furthermore, the levels of circulating nucleosomes on admission were significantly correlated with the inflammatory response and organ dysfunction in sepsis. Meanwhile, a trend that the admission levels of circulating nucleosomes in non-survivors were higher than those in survivors was observed.Conclusions:Serum circulating nucleosomes has a predictive value for sepsis and organ dysfunction, and may serve as a candidate biomarker for diagnosis/prognosis of sepsis. Further studies are warranted to confirm the present findings. Sepsis, a systemic inflammatory reaction to infections, is associated with substantial morbidity and mortality. During the infection, the innate immune response plays a crucial role in satisfactory host resolution of infection. As we know, neutrophils are important effector cells in the immune system’s arsenal against invading pathogens. In addition to the phagocytosis and killing microbes upon phagolysosomal fusion, activated neutrophils release granule proteins and chromatin to form fibres called neutrophil extracellular traps (NETs). NETs are abundant at inflammatory sites and can efficiently bind, engulf, and kill both Gram-positive and Gram-negative bacteria, fungi and parasites extracellularly. Interestingly, some pathogens are captured, but not killed in NETs through DNases damaging the structure of the NETs as well as an antiphagocytic capsule. During sepsis, both neutrophils and platelets are actived, and together they cooperate to fomating NETs, which trapping bacterials in the vasculature and also inducing damage in tissue.更多还原