【作者】 李欢；

【导师】 张平安；

【作者基本信息】 武汉大学 ， 临床检验诊断学， 2022， 硕士

【摘要】 第一部分 GDF15对脓毒症患者的诊断与预后价值分析研究背景与目的:脓毒症是医院重症医学科发病率和死亡率非常高的疾病,脓毒症的早期诊断对提高患者预后有非常重要的意义。目前应用于脓毒症的血清生物标志物非常多,但均具有一定的局限性,多种生物标志物联合才能够更为严谨准确的对脓毒症进行早期诊断辅助临床医生用药治疗和判断预后。生长分化因子15(GDF15)是一种应激性分泌型蛋白,有研究指出脓毒症患者血清GDF15水平显著升高,但未进行GDF15在脓毒症临床应用中的系统评估。本部分研究主要目的是分析GDF15与脓毒症器官损伤的相关性,同时探讨GDF15联合其它炎症指标对脓毒症的诊断价值,以及GDF15水平的动态监测对病情预后判断的临床价值。研究方法:1.收集2020年7月至2021年4月于武汉大学人民医院就诊的脓毒症组患者132例,非脓毒症组93例和对照组95例,并对脓毒症患者完成序贯器官衰竭评分(SOFA)。将脓毒症患者根据第三版脓毒症专家共识分为脓毒症组和休克组;另根据脓毒症患者入院28天生存情况分为存活组和死亡组。2.采用酶联免疫吸附试验(ELISA)检测所有研究对象的GDF15和白介素10(IL-10)水平。3.采用全自动血细胞分析仪、全自动化学发光免疫分析仪和循环增强荧光发光仪分别检测所有研究对象的血常规指标、降钙素原(PCT)和白介素-6(IL-6)水平。4.采用全自动特定蛋白分析仪检测所有研究对象的C反应蛋白(CRP)和血清淀粉样蛋白A(SAA)水平。5.采用全自动生化分析仪、全自动凝血分析仪和全自动化学发光分析仪分别检测脓毒症患者的生化指标、凝血指标和心肌损伤指标。研究结果:1.脓毒症组血清GDF15水平显著高于非脓毒症组和对照组(P值均小于0.05)。2.脓毒症组血清GDF15水平与PCT水平和SOFA评分均存在相关性(相关系数分别为0.3939和0.5786,P值均小于0.05)。3.脓毒症组血清GDF15水平与肝脏、肾脏和心肌损伤指标均具有相关性,且与凝血指标和细胞因子相关。4.GDF15对脓毒症早期诊断ROC曲线的AUC为0.821,联合其它炎症生物标志物及SOFA评分可将AUC提高到0.982。5.脓毒症组和休克组血清GDF15的动态水平比较差异无统计学意义,但脓毒症休克组的血清GDF15水平整体呈现逐渐升高的趋势,而脓毒症组的变化不大。ROC曲线分析显示监测第7天的GDF15水平对鉴别脓毒症休克的价值最高。6.脓毒症死亡组血清GDF15动态水平高于存活组,存活组患者GDF15水平呈现降低的趋势,而死亡组患者GDF15水平呈现升高的趋势。ROC曲线分析结果显示,监测第7天的GDF15水平对脓毒症预后判断的价值最高。研究结论:1.脓毒症患者血清GDF15水平异常升高,其升高水平可能与疾病严重程度相关。2.脓毒症患者血清GDF15水平可能与器官损伤,凝血功能异常和炎症反应密切相关。3.GDF15对脓毒症具有较好的早期诊断价值,与其它脓毒症生物标志物联合可进一步提高对脓毒症的诊断效能。4.动态监测血清GDF15水平或许可以提示脓毒症的病情和预后。第二部分 GDF15对巨噬细胞炎症反应及吞噬杀菌功能的调节作用研究背景与目的:病原体入侵机体后,若不能及时清除病原体和控制炎症,极有可能导致炎症反应放大而损伤正常的组织细胞。巨噬细胞是病原体入侵后最主要的效应细胞,在负责病原体清除的同时,也发挥着重要的抗炎功能,当炎症反应失控时,巨噬细胞则会激发促炎效应而导致机体损伤。因此脓毒症发生时,巨噬细胞的功能不可忽视。本课题第一部分结果显示,GDF15与各器官损伤紧密相关,提示GDF15参与了脓毒症的发生发展。本部分研究旨在从细胞层面分析GDF15对巨噬细胞炎症反应的调节作用,并进一步探讨GDF15对巨噬细胞吞噬和杀菌功能的影响,明确GDF15在脓毒症的发生发展中的作用。研究方法:1.培养人髓系白血病单核细胞(THP-1)和小鼠单核巨噬细胞白血病细胞(RAW264.7)。THP-1细胞为悬浮细胞,实验前先用佛波酯(PMA)刺激贴壁。脂多糖(LPS)刺激细胞模拟体外炎症模型后,分别采用ELISA法和Western blot法检测培养基上清和胞内GDF15的表达情况。2.采用GDF15重组蛋白(rGDF15)对THP-1和RAW264.7细胞预处理0.5 h,再以LPS刺激6 h,使用流式细胞仪检测培养基上清中IL-6和肿瘤坏死因子α(TNF-α)的水平。提取细胞总RNA逆转录后采用实时荧光定量PCR法检测巨噬细胞极化标志物CD80和CD163的表达水平。3.采用庆大霉素保护试验评估巨噬细胞的吞噬和杀菌功能,实验组的处理条件为rGDF 15,对照组为PBS。研究结果:1.与空白对照组对比,LPS刺激后THP-1和RAW264.7细胞培养基上清和胞内的GDF15水平均上调。2.与空白对照组对比,LPS刺激后的细胞因子水平均显著升高;与LPS组对比,rGDF15预处理后细胞因子水平均有下调趋势。3.LPS刺激后巨噬细胞M1型极化标志物CD80 mRNA表达水平升高,而采用rGDF15预处理,CD80 mRNA水平有所下降,而M2型极化标志物CD163 mRNA水平则升高。4.rGDF15处理组巨噬细胞的吞噬和杀菌功能均高于PBS处理组。研究结论:1.LPS的刺激可以提高GDF15的表达与分泌。2.GDF15可以抑制巨噬细胞炎症状态下细胞因子的释放。3.GDF15可以抑制巨噬细胞M1型极化,并促进M2型极化。4.GDF15或许能够促进巨噬细胞的吞噬和杀菌功能。第三部分 GDF15对炎症信号通路JAK1/STAT3和NF-kB p65核转位的调控作用研究背景与目的:本研究第二部分已证实GDF15可以下调巨噬细胞的炎症反应并抑制M1型极化,但涉及的信号通路分子尚不清楚,而信号通路上的关键分子经常作为临床疾病的治疗靶点,所以本部分拟进一步探讨GDF15在脓毒症中发挥保护功能时调控的信号通路,为GDF15尽早应用于临床提供更有力的证据。与炎症反应密切相关的信号通路非常多,其中Janus激酶1/信号转导和转录激活因子3(JAK1/STAT3)以及核因子κB(NF-κB)信号通路是调控脓毒症的炎症反应的经典通路。炎症反应的出现往往伴随了 JAK1/STAT3和NF-κB信号通路的激活,JAK1/STAT3激活后又会介导炎性介质的进一步释放,使得炎症级联反应放大。当NF-κB信号激活后,其亚基p65会从细胞质进入细胞核(也即核转位),随后与核内DNA特异序列相结合,促进多种基因的转录,其中就包括各种参与炎症反应的细胞因子。已有研究提出,抑制NF-κB p65核转位可以减轻脓毒症炎症反应和器官损伤。而GDF15对上述两个关键信号通路的调控作用尚不清楚,本部分内容旨在对第二部分研究结果进行深入分析,探讨GDF15发挥抗炎作用是否与炎症信号通路JAK1/STAT3磷酸化水平和NF-κB p65核转位水平降低相关。研究方法:1.培养THP-1和RAW264.7细胞,THP-1细胞为悬浮细胞,实验前先用PMA刺激贴壁。采用rGDF15对THP-1和RAW264.7细胞预处理0.5 h,再以LPS 刺激 6 h。2.提取细胞总蛋白,应用Western blot法检测JAK1/STAT3信号通路的磷酸化水平。3.采用细胞免疫荧光法测定巨噬细胞NF-κB p65的核转位情况。4.分离细胞核蛋白,应用Western blot法检测胞核磷酸化NF-κB p65的表达水平。研究结果:1.与空白对照组对比,LPS组JAK1/STAT3的磷酸化水平显著升高,而rGDF15的预处理可以有效抑制JAK1/STAT3的磷酸化。2.rGDF 15的预处理下调了 LPS刺激后NF-κB p65的核转位水平。3.rGDF 15预处理抑制了 LPS刺激后胞核磷酸化NF-κB p65的表达。研究结论:GDF15可能是通过抑制JAK1/STAT3磷酸化和NF-κB p65的核转位水平减轻炎症反应。更多还原

【Abstract】 Part 1 Diagnostic and Prognostic Value of GDF15 in Patients with SepsisBackground and Objective:Sepsis is a disease with high morbidity and mortality in the intensive care unit(ICU)of hospitals.Early diagnosis of sepsis is of great significance for improving the prognosis of patients.At present,there are many serum biomarkers used in sepsis,but they all have certain limitations.Only by combining a variety of biomarkers can we make a more rigorous and accurate early diagnosis of sepsis and assist clinicians in medication treatment and prognosis.Growth differentiation factor 15(GDF15)is a stress secretory protein.Some studies have pointed out that the level of serum GDF15 in patients with sepsis is significantly increased,but there is no systematic evaluation of the clinical value of GDF15 in sepsis.The main purpose of this study is to analyze the correlation between GDF15 and organ injury in sepsis,and to further explore the diagnostic value of GDF15 combined with other inflammatory indexes in sepsis and the clinical value of dynamic monitoring of GDF15 in judging the condition and prognosis of sepsis.Methods:1.132 patients in the sepsis group,93 patients in the nonsepsis group and 95 patients in the control group were collected from July 2020 to April 2021 in the Renmin Hospital of Wuhan University.Sequential organ failure score(SOFA)was completed on patients with sepsis.Sepsis patients were further divided into sepsis group and septic shock group according to The Third International Consensus Definitions for Sepsis and Septic Shock(Sepsis-3);In addition,according to the 28-day survival of patients with sepsis,we divided them into survival group and death group.2.The serum levels of GDF15 and interleukin-10(IL-10)were tested by enzymelinked immunosorbent assay(ELISA).3.The blood routine indexes,procalcitonin(PCT)and interleukin-6(IL-6)levels of all subjects were detected by automatic blood cell analyzer,automatic chemiluminescence immunoanalyzer and cycle enhanced fluorescence analyzer,respectively.4.The levels of C-reactive protein(CRP)and serum amyloid A(SAA)were detected by automatic specific protein analyzer.5.The biochemical indexes,coagulation indexes and myocardial injury indexes of patients with sepsis were detected by automatic biochemical analyzer,automatic coagulation analyzer and automatic chemiluminescence analyzer,respectively.Results:1.The serum levels of GDF15 in sepsis group were significantly higher than those in nonsepsis group and control group(P<0.05).2.The serum levels of GDF15 were correlated with PCT and SOFA in patients with sepsis(the correlation coefficients were 0.3939 and 0.5786,respectively,P<0.05).3.The serum levels of GDF15 in sepsis group were correlated with liver,kidney and myocardial injury indexes,as well as coagulation indexes and cytokines.4.The AUC of ROC curve of early diagnosis of sepsis by GDF15 was 0.821,while combined with other inflammatory biomarkers and SOFA score,the AUC could be increased to 0.982.5.There was no significant difference in the dynamic serum levels of GDF15 between sepsis group and sepsis shock group,but the serum levels of GDF15 in sepsis shock group increased gradually,while there was little change in sepsis group.ROC curve analysis showed that monitoring the levels of GDF 15 on day 7 had the highest value in differentiating septic shock.6.The dynamic serum levels of GDF 15 in sepsis death group were higher than those in survival group.The serum levels of GDF 15 in survival group decreased,while the serum levels of GDF 15 in death group increased.ROC curve analysis showed that monitoring the levels of GDF 15 on day 7 had the highest value in judging the prognosis of sepsis.Conclusions:1.The levels of serum GDF 15 in patients with sepsis were abnormally elevated,which may be related to the severity of the disease.2.The levels of serum GDF 15 in patients with sepsis may be closely related to organ damage,abnormal coagulation function and inflammatory response.3.GDF 15 had good value in early diagnosis of sepsis.And combined with other sepsis biomarkers,it could further improve the diagnostic efficiency of sepsis.4.Dynamic monitoring of serum GDF 15 levels may indicate the condition and prognosis of sepsis.Part 2 Regulation of GDF15 on inflammatory response and phagocytic and bactericidal function of macrophagesBackground and Objective:When pathogens invade the body,if the pathogens cannot be cleared and inflammation cannot be controlled in time,it is very likely to lead to amplification of inflammatory response and damage to normal tissues and cells.Macrophages are the most important effector cells after pathogen invasion.While responsible for pathogen clearance,they also play an important anti-inflammatory function.When the inflammatory response is out of control,macrophages will stimulate the proinflammatory effect and lead to body damage.Therefore,when sepsis occurs,the function of macrophages can’t be ignored.The results of the first part of this study show that GDF15 is closely related to the damage of various organs,suggesting that GDF15 is involved in the occurrence and development of sepsis.This part of the study aims to analyze the regulatory effect of GDF15 on the inflammatory response of macrophages from the cellular level,and to further explore the effect of GDF15 on the phagocytosis and bactericidal function of macrophages,and then to clarify the role of GDF15 in the occurrence and development of sepsis.Methods:1.Human myeloid leukemia monocytes(THP-1)and mouse monocyte macrophage leukemia cells(RAW264.7)were cultured.THP-1 cells were suspended cells,and phorbol ester(PMA)was used to stimulate adherence before the experiment.After lipopolysaccharide(LPS)stimulated cells to simulate in vitro inflammation model,the expression of GDF15 in medium supernatant and cells was detected by ELISA and Western blot,respectively.2.The THP-1 and RAW264.7 cells were pretreated with GDF15 recombinant protein(rGDF15)for 0.5 h,and then stimulated with LPS for 6 h.Flow cytometry was used to detect the levels of IL-6 and tumor necrosis factor alpha(TNF-α).Total cellular RNA was extracted,and real-time fluorescent quantitative PCR was used to detect the expression levels of macrophage polarization markers(CD80 and CD 163)after reverse transcription.3.The phagocytic and bactericidal functions of macrophages were evaluated by the gentamicin protection test.The treatment condition of the experimental group was rGDF15,and the control group was PBS.Results:1.Compared with the blank group,the levels of GDF15 in the culture supernatant and intracellular of THP-1 and RAW264.7 cells were up-regulated after LPS stimulation.2.Compared with the blank group,the cytokine levels of the LPS treatment group were significantly increased;compared with the LPS treatment group,the cytokine levels after rGDF 15 pretreatment had a downward trend.3.After LPS stimulation,the expression mRNA level of M1 polarization marker CD80 in macrophages increased,while pretreatment with rGDF 15 decreased the mRNA level of CD80,which was accompanied by an increase in the mRNA level of M2 polarization marker CD 163.4.The phagocytic and bactericidal functions of macrophages in the rGDF 15 treatment group were higher than those in the PBS treatment group.Conclusions:1.The stimulation of LPS could increase the expression and secretion of GDF15.2.GDF15 could inhibit the release of cytokines in the inflammatory state of macrophages.3.GDF15 could restrain M1 type polarization and facilitate M2 type polarization of macrophages.4.GDF15 may improve the phagocytosis and bactericidal function of macrophages.Part 3 Regulation of GDF15 on Inflammatory Signaling Pathways JAK1/STAT3 and NF-κB p65 Nuclear TranslocationBackground and Objective:The second part of this study has confirmed that GDF15 can reduce the inflammatory response of macrophages and inhibit M1 polarization,but the involved signal pathway molecules are not clear.The key molecules in the signal pathway are often used as the therapeutic targets of clinical diseases.Therefore,this part of the study intends to further explore the signal pathway regulated by GDF15 when it plays a protective role in sepsis and to provide more powerful evidence for the early application of GDF15 in clinic.There are many signal pathways closely related to inflammatory response,including Janus kinase 1/signal transducer and activator of transcription 3(JAK1/STAT3)and nuclear factor κB(NF-κB),which are the classical pathway regulating the inflammatory response of sepsis.The emergence of inflammatory response is often accompanied by the activation of JAK1/STAT3 and NF-κB signal pathway.After the activation of JAK1/STAT3,it will mediate the further release of inflammatory mediators and amplify the inflammatory cascade.When NF-κB signal is activated,its subunit p65 will enter the nucleus from the cytoplasm(i.e.,nuclear translocation),and then combine with specific DNA sequences in the nucleus to promote the transcription of related genes,including various cytokines involved in inflammatory response.Studies have proposed that the inhibition of NF-κB p65 nuclear translocation can reduce the inflammatory response and organ damage in sepsis.The regulatory effect of GDF15 on the above two key signal pathways is not clear.This part aims to deeply analyze the results of the second part,and to explore whether the anti-inflammatory effect of GDF15 is related to the decreased levels of phosphorylation of JAK1/STAT3 and nuclear translocation of NFκB p65.Methods:1.THP-1 and RAW264.7 cells were cultured.THP-1 cells were suspended cells,and phorbol ester(PMA)was used to stimulate adherence before the experiment.The THP-1 and RAW264.7 cells were pretreated with rGDF15 for 0.5 h,and then stimulated with LPS for 6 h.2.The total protein was extracted and the phosphorylation level of JAK1/STAT3 signal pathway was detected by Western blot.3.The nuclear translocation of NF-κB p65 in macrophages was measured by cellular immunofluorescence.4.The nuclear protein was isolated and the expression level of nuclear phosphorylated NF-κB p65 was detected by Western blot.Results:1.Compared with the blank control group,the phosphorylation level of JAK1/STAT3 in LPS group was significantly increased,and the pretreatment of rGDF15 could effectively inhibit the phosphorylation of JAK1/STAT3.2.Pretreatment with rGDF15 down regulated the nuclear translocation level of NFκB p65 after LPS stimulation.3.The pretreatment of rGDF15 inhibited the expression of nuclear phosphorylated NF-κB p65 stimulated by LPS.Conclusion:GDF15 may reduce inflammatory response by inhibiting JAK1/STAT3 phosphorylation and nuclear translocation of NF-κB p65.更多还原