脓毒症ARDS病程不同时期肺泡巨噬细胞源性蛋白质组学的变化及其临床意义

发布时间：2018-06-27 01:23

本文选题：ARDS + 脓毒血症　；参考：《中南大学》2014年博士论文

【摘要】：第一部分脓毒症ARDS患者病程不同时期肺泡巨噬细胞蛋白质组学的变化背景与目的急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)是一种肺内过度性、失控性的炎症反应,多种炎症细胞和炎症介质是ARDS发生发展的关键因素。肺泡巨噬细胞(alveolar macrophage, AM)是机体重要的炎性细胞,通过研究在ARDS病程不同时期中AM蛋白质表达的变化,可能为阐明ARDS发病机理提供新的线索和思路。方法以14个继发于脓毒血症的ARDS患者为研究对象,分别于并发中度ARDS后病程的第一天(第一组)及病情明显好转的第五天(第二组)取支气管肺泡灌洗液,从中提取肺泡巨噬细胞,应用双向凝胶电泳(Two dimensional electrophoresis,2DE)和基质辅助激光解析电离飞行时间质谱(Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry, MALDI-TOF-MS)技术筛选鉴定出差异蛋白质。结果两组AM共发现135个显著不同的点,其中87个点容积的变化2.5倍,48个点显示2.5倍的变化。进一步通过串联质谱确定差异蛋白质的身份。最终有35个蛋白质得以确认。与第一组相比较,第二组中表达上调的蛋白质点有21个,表达下调的蛋白质点有14个。结论脓毒症ARDS患者病程不同时期肺泡巨噬细胞蛋白质存在差异性表达。第二部分脓毒症ARDS患者病程不同时期肺泡巨噬细胞源性HSP27及S100A9、TNF-a、IL-1ra表达的差异背景本研究的第一部分选择了继发于脓毒血症的ARDS患者,在病程不同时期取肺泡巨噬细胞进行蛋白质组学分析,研究肺泡巨噬细胞的蛋白质组学的动态变化,通过串联质谱最终有35个蛋白质得以确认,其中表达上调的蛋白质点有21个,表达下调的蛋白质点有14个。本部分研究选择表达下调的蛋白质之一热休克蛋白27(heat shock protein27, HSP27)以及表达上调的蛋白质S100A9、肿瘤坏死因子-a(Tumor Necrosis Factor-a, TNF-a).白细胞介素-1受体拮抗剂(Interleukin-1receptor antagonist, IL-1ra)行Western Blot实验对第一部分结果进行验证。目的探讨脓毒症ARDS患者在病程的不同时期HSP27及S100A9、TNF-a, IL-lra在肺泡巨噬细胞是否有表达及表达是否有差异。方法以5个脓毒症ARDS患者为研究对象,分别于/ARDS发病的第一天及第五天行支气管肺泡灌洗,从中提取肺泡巨噬细胞,采用Western Blot检测ARDS病程不同时期肺泡巨噬细胞HSP27及S100A9、TNF-a、IL-1ra的差异表达。结果TNF-α、IL-1ra、S100A9及HSP27在ARDS患者的肺泡巨噬细胞中均有表达,表达率100%。TNF-α、IL-lra、S100A9在第二组(ARDS病程第5天)比第一组(ARDS病程第1天)表达明显增强：TNF-α表达相对值分别是dayl (0.41±0.03), day5(0.74±0.07) P0.05; IL-lra:dayl (0.26±0.04), day5(0.54±0.06) P0.05; S100A9表达相对值分别是：dayl (0.29±0.02), day5(0.48±0.03) P0.05。而HSP27在第二组(ARDS病程第5天)比第一组(ARDS病程第1天)表达明显减弱,表达相对值分别是dayl(0.60±0.04), day5(0.35±0.02), P0.05。两组间TNF-α、IL-1ra、S100A9及HSP27表达的差异与经过2-DE分析后的蛋白质组学结果一致。结论脓毒症ARDS患者在病程的不同时期HSP27及TNF-α、IL-lra、S100A9在肺泡巨噬细胞均有差异性表达,且与蛋白质组学结果一致。第三部分脓毒症ARDS患者血浆及支气管肺泡灌洗液中TNF-a、IL-1、IL-Ira、IL-10、vWF及RAGE的水平及对ARDS病情发展及预后的评估背景ARDS是以突出的肺部损伤、炎症及微血管通透性增加为特征进而导致弥漫性肺水肿和机械性功能障碍,最终导致呼吸衰竭的一种疾患。ARDS患者的主要死因是由于肺损伤引起的系统性炎症所导致的多器官功能衰竭。在本研究的第一部分获得了在ARDS病程不同时期肺泡巨噬细胞差异性表达的蛋白质。在本研究的第二部分,用Western Blot的研究方法证实ARDS患者在病程的不同时期TNF-a及IL-1ra在肺泡巨噬细胞有差异性表达,且与蛋白质组学结果一致。因此在第一、第二部分研究的基础上,本部分研究选择在ARDS患者血浆及肺泡灌洗液中检测TNF-a、IL-1ra及其他炎症介质研究其对ARDS病情发展及预后的预测价值。目的探讨ARDS患者血浆及支气管肺泡灌洗液(BALF)中TNF-a、IL-1、IL-1ra、IL-10、晚期糖基化终末产物受体(receptor for advanced glycation end products,RAGE)、von Villebrand因子(vWF)对ARDS病情发展及预后的预测作用。方法研究对象选择脓毒症ARDS患者54例,所有研究对象收住ICU时均处于病情的急性期,并发ARDS24小时以内,在收住ICU病房的第1天6小时内立即收集外周血液标本和肺泡灌洗液(BALF)标本。用酶联免疫吸附试验测定细胞因子TNF-a、IL-1、IL-1ra、IL-10、vWF及RAGE的浓度以研究这些参数对ARDS发展及预后的预测作用。研究对象分为三组：A组(n=20)为中度ARDS患者组；B组(n=17)为轻度ARDS患者组,该组患者在48小时内发展为中度ARDS;C组(n=17)为轻度ARDS患者组,该组患者未发展为中度ARDS。结果轻度ARDS患者血浆及灌洗液中TNF-a、IL-1、及RAGE、vWF均是病情进展恶化很好的阴性预测值,其中血浆中以上各指标的阴性预测值为83%-95%,灌洗液中88%-94%。血浆中IL-1及vWF的水平和灌洗液中IL-1的水平与死亡正相关,随着IL-1及vWF的水平增高,死亡风险增高。血浆IL-10、IL-1ra水平与死亡呈负相关,血浆IL-10、IL-lra水平越高,死亡风险越低。结论血浆及肺泡灌洗液中的细胞因子TNF-a、IL-1、IL-1ra、IL-10及RAGE、vWF对脓毒症ARDS患者病情进展及预后有一定的预测作用。
[Abstract]:Proteomics changes of alveolar macrophages in patients with ARDS after first partial sepsis

BACKGROUND & OBJECTIVE Acute respiratory distress syndrome ( ARDS ) is a key factor in the development of ARDS after acute respiratory distress syndrome ( ARDS ) .

Methods The alveolar macrophages were extracted from 14 ARDS patients who were secondary to sepsis . The alveolar macrophages were extracted from alveolar macrophages by two dimensional electrophoresis ( 2DE ) and matrix assisted laser desorption / ionization time of Flight Mass Spectrometry ( MALDI - TOF - MS ) .

Results There were 135 significant differences between the two groups . The changes of 87 points were 2.5 times and 2.5 times . The identity of the difference protein was determined by tandem mass spectrometry . The final 35 proteins were confirmed . There were 21 protein spots up - regulated in the second group compared with the first group , and 14 of the down - regulated protein spots were expressed .

Conclusion There is different expression of alveolar macrophage protein in patients with sepsis ARDS .

The difference of expression of HSP27 , TNF - a and IL - 1ra in alveolar macrophages during different stages of ARDS in patients with second partial sepsis

Background : The first part of this study selected ARDS patients who were secondary to sepsis , studied the proteomic analysis of alveolar macrophages in different stages of the course of the disease , studied the dynamic changes of protein groups in alveolar macrophages , and confirmed that there were 21 protein spots up - regulated by tandem mass spectrometry . The first part of IL - 1receptor antagonist ( IL - 1ra ) was verified by Western Blot .

Objective To investigate the expression and expression of HSP27 ( HSP27 ) , TNF - a ( TNF - a ) and interleukin - lra ( IL - lra ) in alveolar macrophages in ARDS patients .

Methods The alveolar macrophages were extracted from the first day and the fifth day of the onset of ARDS in five sepsis ARDS patients , and the difference of HSP27 in the alveolar macrophages and the levels of TNF - a and IL - 1ra in the alveolar macrophages in different stages of ARDS were detected by Western Blot .

Results The relative values of TNF - 伪 , IL - 1ra , S100 A9 and HSP27 were significantly increased in the second group ( 0 . 41 卤 0 . 03 ) , day5 ( 0 . 74 卤 0 . 07 ) P 0 . 05 ; IL - lra : dayl ( 0.26 卤 0.04 ) , day5 ( 0.54 卤 0.06 ) P 0 . 05 ; the relative values of HSP27 expression were dayl ( 0 . 60 卤 0.04 ) , day5 ( 0 . 35 卤 0.02 ) , P 0 . 05 .

Conclusion HSP27 and TNF - 伪 , IL - lra and S100 A9 were differentially expressed in alveolar macrophages in ARDS patients at different stages and were consistent with the results of Proteomics .

The levels of TNF - a , IL - 1 , IL - Ira , IL - 10 , VWF and RAGE in plasma and bronchoalveolar lavage fluid in patients with third partial sepsis ARDS and the evaluation of the development and prognosis of ARDS

In the second part of this study , we studied the expression of TNF - a , IL - 1ra and other inflammatory mediators in the plasma and alveolar lavage fluid of ARDS patients , and the predictive value of TNF - a , IL - 1ra and other inflammatory mediators on the development and prognosis of ARDS patients was studied .

Objective To investigate the prognostic significance of TNF - a , IL - 1 , IL - 1ra , IL - 10 , advanced glycosylation end products ( RAGE ) and von Wilk factor ( VWF ) on the development and prognosis of ARDS patients .

Methods 54 cases of ARDS patients with sepsis were studied . All the subjects were in the acute stage of the condition in the ICU , and the peripheral blood samples and BALF samples were collected immediately within 6 hours of the first day of the intensive care unit . The concentrations of cytokines TNF - a , IL - 1 , IL - 1ra , IL - 10 , von Wilvand RAGE were measured by enzyme - linked immunosorbent assay . The results were divided into three groups : group A ( n = 20 ) as the group of moderate ARDS patients ;
Group B ( n = 17 ) was a mild ARDS group and the group developed into moderate ARDS within 48 hours ; Group C ( n = 17 ) was mild ARDS patients and the group did not develop into moderate ARDS .

Results TNF - a , IL - 1 , IL - 1 , IL - 1 , IL - 1 and IL - 1 levels in plasma and lavage fluid of patients with mild ARDS were negative predictive values . The levels of IL - 1 , IL - 1ra and IL - 1 in plasma were negatively correlated with death . The higher the levels of IL - 10 and IL - 1ra in plasma , the higher the level of IL - 10 and IL - lra , the lower the risk of death .

Conclusion TNF - a , IL - 1 , IL - 1ra , IL - 10 and RAGE in plasma and alveolar lavage fluid can predict the progression and prognosis of ARDS patients .
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R459.7

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