【作者】 刘洋；

【导师】 程明亮；

【作者基本信息】 苏州大学 ， 传染病学（专业学位）， 2022， 博士

【摘要】 研究背景:急性肝功能衰竭(acute hepatic failure,ALF)发生时肝细胞呈一过性大块或亚大块坏死,或桥接坏死,伴肝细胞严重变性。其病因多样,机制不明,尚无特效药物,肝移植是目前唯一有效治疗方法,死亡率极高。研究发现,全身炎症反应综合征(systemic inflammatory reaction syndrome,SIRS)的激活与ALF的发生与进展密切相关,以促炎介质激活如半胱天冬酶-1(Caspase-1)、核因子κB(nuclear factorkappa-B,NF-κB)和炎症介质过度释放如白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子(tumor necrosis factor,TNF-α)等为特征,使肝细胞受到炎症刺激,引发复杂的免疫反应,最终发生程序性死亡。细胞焦亡(Pyroptosis)是一种和炎症密切相关的新型细胞程序性死亡,表现为细胞破裂后释放细胞内容物并激活强烈的炎症反应,是机体重要的天然免疫反应,在抗击感染中发挥重要作用。有研究发现ALF的病程中伴随着细胞焦亡的发生,但焦亡对ALF的机制研究尚少。本研究旨在利用高通量芯片数据筛选出对ALF发病机制研究或临床治疗有启发的新目标蛋白并加以实验验证。差异列表中,Eva-1同系物A(eva-1 homologue A,EVA1A)的表达与多种肝脏疾病密切相关。EVA1A也称为跨膜蛋白166(transmembrane protein 166,TMEM166)或序列相似性176家族A(FAM176A),是一种溶酶体和内质网相关蛋白。研究发现,EVA1A通过抑制肝癌细胞增殖和分化来体现抑癌特性;EVA1A的高表达可抑制非活动性乙肝携带者的病毒复制;EVA1A被视为介导自噬和细胞凋亡的一种调节剂,和细胞程序性死亡密切相关。细胞程序性死亡是ALF进程中贯穿始末的核心事件。因此,深入解析EVA1A和ALF的关系以及EVA1A对ALF中细胞程序性死亡的调控有助于了解ALF的发病机制或找到治疗的新靶点。另外,从miRNAs数据库预测到EVA1A的表达受到上游miR-125b-5p的靶向抑制。miR-125b-5p通常在巨噬细胞中高表达,可促进巨噬细胞表达更多的共刺激分子并激活靶细胞,调节靶细胞增殖、凋亡和分化等功能。由此,深入研究miR-125b-5p/EVA1A信号轴调节肝细胞的炎症及焦亡,通过抑制肝细胞的促炎蛋白活性或阻断焦亡关键分子可能是治疗ALF的新方向。库普氏细胞(kupffer cells,KCs)又称为常驻肝巨噬细胞,位于肝窦内表面,是肝细胞受到炎症刺激或发生死亡时最先被激活的免疫细胞,通过分泌各种细胞因子和介质发挥免疫调节功能,在维持肝内环境稳态中起着核心作用。外泌体(exosome,Exo)是细胞分泌的胞外小囊泡(extracellular vesicles,EVs),其大小为30-150mm,具有双层膜结构和茶托状形态,含有丰富的内含物(包括核酸、蛋白和脂质等),广泛存在于体液中,参与细胞间物质运输与信息传递,调控机体免疫应答、抗原递呈、细胞分化、抗肿瘤侵袭等多种生理病理活动,医学应用前景广阔。研究发现,已有多种miRNAs广泛参与了调节细胞程序性死亡的步骤和介质。KCs可通过外泌体携带microRNAs(miRNAs)的方式在不同类型细胞间实现信息交互和通讯,进而对受体细胞发挥调节作用,因此,探讨ALF中KCs能否通过外泌体携带miRNAs和新靶标来调节肝细胞的炎症水平或焦亡,不仅对于ALF的发病机制研究具有参考价值,也将为研宄ALF的有效治疗方法提供新的思路和方向.研究目的:通过生信学方法筛选在ALF中表达差异明显的靶标,预测上游miRNA,通过生物学技术验证两者与急性肝衰竭的关系;证实ALF发病机制中有肝细胞炎症和焦亡的参与;探讨KCs来源的外泌体通过miR-125b-5p/EVA1A轴对ALF中肝细胞的炎症和焦亡实现跨细胞调控。研究方法:第一部分:从GEO数据库中筛选出3个乙肝相关性ALF数据集((GSE14668、GSE38941、GSE62037);利用R语言软件筛选差异基因列表;通过GO分析进行可视化;利用STRING数据库构建PPI网络并最终鉴定出目标基因EVA1A;通过TargetScan、miRDB、miRWalk3个数据库取交集预测EVA1A受miR-125b-5p调控。收集5例ALF患者肝组织和血标本,利用D-Galn/LPS构建L02细胞株炎症模型和C57BL/6小鼠ALF模型,通过ELISA、qRT-PCR、Westem blot等方法从组织、细胞、动物三个层面检测IL-1β,TNF-α,Caspase-1、NF-κB p65及EVA1A的水平,QT-PCR检测miR-125b-5p的表达;双荧光素酶报告基因实验验证miR-125b-5p对EVA1A的靶向负调控关系;通过转染agomiR和antagomiR、转染si EVA1A和质粒验证miR-125b-5p/EVA1A轴对NF-κBp65的调控,进而影响对炎症因子和促炎介质的水平。第二部分:从D-Galn(20mmol/L)/LPS(100μg/mL)浓度环境下的KCs培养上清中分离Exo,并经透射电子显微镜、纳米颗粒跟踪分析及WesternBlot鉴定;通过荧光标记检验AML12细胞对KCs-Exo的摄取,并检验D-Galn/LPS诱导的KCs-Exo通过miR-125b-5p/EVA1A轴对AML12细胞的焦亡关键分子GSDMD的影响。在构建的ALF小鼠模型中通过尾静脉注射KCs-Exo和antagomiR-125b-5p验证KCs-Exo可携带miR-125b-5p通过调控EVA1A调控肝脏中焦亡的发生。最后在ALF患者肝组织和外周血验证焦亡的存在和外泌体的分泌增多。研究结果:第一部分:1.通过生物信息学方法筛选出ALF相关的表达差异基因集,通过注释、可视化分析得知这些差异基因主要参与免疫反应、炎症、急性应激、氧化还原过程、补体激活调控等生物学功能,并在细胞外泌体、细胞间隙等场所富集;生物代谢通路、PPAR信号途径等受到显著调节;从表达下调最明显的27个基因中将EVA1A作为目标蛋白;预测EVA1A受上游miR-125b-5p负向调控。2.qRT-PCR及双荧光素酶报告基因实验验证了miR-125b-5p和EVA1A的靶向负调控;在D-Galn/LPS诱导的L02细胞模型中,IL-1β、TNF-α、Caspase-1以及NF-κBp65高表达,miR-125b-5p显著上调而EVA1A显著下调;改变miR-125b-5p表达量影响EVA1A和NF-κB p65的表达,进而调节L02细胞的炎症水平:上调miR-125b-5p则显著抑制EVA1A水平并提高NF-κB p65表达,而下调miR-125b-5p则可提高EVA1A的水平并抑制NF-κB p65对炎症的促进作用。3.成功构建ALF小鼠模型,通过苏木精-伊红染色、ALT、AST及INR的测定进行模型验证;观察到小鼠肝组织和血清中IL-1β、TNF-α、Caspase-1的表达上调;经尾静脉注射agomiR-125b-5p(激动剂)和antagomiR-125b-5p(抑制剂)观察到肝组织出现形态学改变、炎症因子和促炎介质的表达升高;验证miR-125b-5p通过靶向抑制EVA1A促进NF-κB p65的表达,进而加剧小鼠肝脏炎症。第二部分:1.通过透射电子显微镜、纳米颗粒跟踪分析及Westem blot的结果证实实验操作可稳定分离出KCs-Exo;在PBS和D-Galn/LPS环境下均可促进KCs-Exo分泌增多,且有显著组间差异;标记于荧光染料的KCs-Exo可被AML12细胞摄入,并导致AML12细胞内成熟miR-125b-5p水平升高;通过agomiR-125b-5p转染或Exo-D-Galn/LPS处理后均可导致AML12细胞中EVA1A荧光素酶的活性,Western blot检测EVA1A的蛋白表达明显下降;转染agomiR-125b-5p或者直接摄入KCs-Exo均能促进GSDMD-N的表达,说明焦亡发生增多,而无论抑制miR-125b-5p还是过表达pcEVA1A,都能抑制GSDMD-N的升高。2.Exo-D-Galn/LPS组血清ALT、AST水平升高,IL-1β、Caspase-1细胞因子浓度升高;Westemblot结果显示,GSDMD的表达增多、EVA1A表达减少;过表达EVA1A后GSDMD的活化被抑制,而这种抑制作用可以被Exo-D-Galn/LPS阻断。3.通过Western blot检测ALF患者体内IL-1 1 1β、Caspase-1水平升高,GSDMD-N的水平也升高,而EVA1A水平下降;ALF患者外泌体的特异蛋白表达和miR-125b-5p的水平较正常人明显升高。研究结论:1.在ALF或炎症过程中,miR-125b-5p/EVA1A信号轴可通过上调NF-κBp65的表达,促进炎症介质的释放,从而加重肝脏的炎症损伤.2.KCs来源的外泌体可通过miR-125b-5p/EVA1A轴上调肝细胞内GSDMD的表达,进而促进肝细胞焦亡。更多还原

【Abstract】 Background:Acute liver failure(acutehepaticfailure,ALF)is a severe liver injury caused by many factors,which leads to serious disturbance or decompensation of liver synthesis,detoxification,excretion and biotransformation within 2 weeks,and clinical symptoms such as coagulation disorder,jaundice,hepatic encephalopathy and ascites.Lack of specific drugs in treatment,liver transplantation is no other effective treatment at present.However,the shortage of organs or the inability to identify the underlying causes affect the prognosis and clinical management,and the mortality rate is extremely high.The pathogenesis of ALF has not been fully elucidated.Studies have found that the activation of systemic inflammatory response syndrome(SIRS)is closely related to the occurrence and progression of ALF,characterized by activation of pro-inflammatory mediators such as Caspase-1,nuclear factor kappa B(NF-κB)and excessive release of inflammatory mediators such as interleukin-1 β(IL-1 β)and tumor necrosis factor(tumor necrosis factor-α,TNF-α).The hepatocytes are stimulated by inflammation,which leads to complex immune response and eventually programmed death.Cell scorch death(Pyroptosis)is anew type of programmed cell death closely related to inflammation,which is characterized by the release of cellular contents and activation of strong inflammatory response after cell rupture.It is an important innate immune response and plays an important role in the fight against infection.Some studies have found that the course of ALF is accompanied by the occurrence of cell death,but there are few studies on the mechanism ofALF.The purpose of this study is used by high-throughput chip data to screen and validate new target proteins that have implications for the pathogenesis or clinical treatment of ALF.Among the most differentially expressed genes,the expression of Eva-1 homologue A (eva-1homologueA,EVA1A)is closely linked to a variety of liver diseases.EVA1 A,also known as transmembrane protein 166 or sequence similarity family A(FAM176A),is a lysosome and endoplasmic reticulum related protein.Studies have found that EVA1 A can inhibit cancer by inhibiting the proliferation and differentiation of liver cancer cells;the high expression of EVA1 A can inhibit virus replication in inactive hepatitis B carriers;EVA1A is regarded as a regulator of autophagy and apoptosis,which is closely related to programmed cell death.Programmed cell death is at the heart event throughout the process of ALF.Therefore,in-depth analysis of the relationship between EVA1 A and ALF and the regulation of EVA1 A on programmed cell death in ALF is helpful to understand the pathogenesis of ALF or find a new target for treatment.In addition,it is predicted from the miRNAs database that the expression of EVA1 A is targeted by upstream miR-125b-5p1MiR-125b-5p is usually highly expressed in macrophages,which can promote macrophages to express more conciliatory molecules and activate target cells,regulating target cell proliferation,apoptosis and differentiation.Therefore,in-depth study of miR-125b-5p/EVA1 A signal axis regulating hepatocyte inflammation and scorch death,by inhibiting the pro-inflammatory protein activity of hepatocytes or blocking the key molecules of scorching death may be a new direction of treatment of ALF.Kup ffer cells(KCs),also known as resident hepatic macrophages,located on the inner surface of the hepatic sinusoid are the first immune cells to be activated when hepatocytes are stimulated by inflammation or death.They play a central role in maintaining the homeostasis of the live environment by secreting various cytokines and mediators:Exocrine bodies(exosomes,Exo)are extracellular vesicles(extracellularvesicles,EVs)secreted by cells,with a size of 30-150 nm,bilayer membrane structure and saucer-like shape,rich in inclusions(including nucleic acids,proteins and lipids,etc.),widely exist in body fluids,particip ate in intercellular material transport and information transmission,regulate a variety of physiological and pathological activities,such as immune response,antigen presentation,cell differentiation,anti-tumor invasion and so on.There are broad prospects for therapeutic application.Studies have found that a variety of miRNAs have been widely involved in the regulation of programmed cell death steps and mediators.KCs can realize information exchange and communication between different types of cells by exosome carrying microRNAs(miRNAs),and then regulate receptor cells.Therefore,to explore whether KCs in ALF can regulate hepatocyte inflammation or death by exosome carrying miRNAs and new targets will not only have reference value for the study of the pathogenesis of ALF,but also provide new ideas and directions for the study of effective treatment of ALFObjective:Using high-throughput chip data,new target proteins and their upstream miRNA were screened by bioinformatics method for the study of ALF mechanism or clinical treatment,and the negative regulation relationship between them was verified by experiments;it was confirmed that hepatocyte inflammation and scorch death were involved in the pathogenesis of ALF,to explore the transnational regulation of hepatocyte inflammation and scorch death in ALF by exocrine from KCs through the miR-125b-5p/EVA1 A axis.Method:Part Ⅰ:Three hepatitis B related ALF datasets(GSE14668,GSE38941,GSE62037)were screened from the GEO database;the list of differential genes was screened by R language software,the visualization was carried out by GO analysis;the PPI network was constructed by STRING database and the target gene EVA1A;was finally identified by TargetScan,miRDB and miRWalk3 databases to predict that EVA1 A was regulated by miR-125b-5p.Liver tissue and blood samples from 5 patients with ALF were collected.L02 cell line inflammatory model and C57BL/6 mice ALF model were constructed by D-GalN/LPS.The levels of IL-1 β,TNF-α,Caspase-1,NF-κB p65 and EVA1 A were detected by ELISA,qRT-PCR and Western blot,and the expression of miR-125b-5p was detected by QT-PCR.Double luciferase reporter gene assay verified the negative targeting regulation ofmiR-125b-5p on EVA1 A,and theregulation of miR-125b-5p/EVA1 A axis on NF-κB p65 was verified by transfection of agomiR and antagomiR,siEVA1 A and plasmid,which further affected the level ofinflammatory factors and pro-infl ammatory mediators.PartⅡ:Mice KCs were extracted and cultured,and exo somes of KCs origin were extracted and identified.The AML12 cell line was selected to construct a cellular inflammation model,and the levels of inflammation-related factors,the expression of mi R-125b-5p/EVA1 A axis,and the expression of GasderminD(GSDMD),akey protein of scorch death,were examined in AML12 cells to further analyze the effect of KCs-derived exosomes carrying miR-125b-5p in interfering with ALM12 under inflammation cells and thus affect the expression of GSDMD,which mediates the scorch death phenotype.We investigated the effects of KCs-derived exosomes on inflammation-related factors and miR-125b-5p and the regulation of GSDMD in mice ALF modelResult:Part Ⅰ:1.ALF-related differential gene sets were screened by bioinformati cs methods and annotated and visual analysis showed that these differential genes were mainly involved in biological functions such as immune response,inflammation,acute stress,redox process,complement activation regulation and so on,and were enriched in exocrine bodies and intercellular spaces,while biological metabolic pathways and PPAR signal pathways were significantly regul ated.EVA1 A was taken as the target protein from the 27 genes with the most down-regulated expression,and it was predicted that EVA1 A was negatively regulated by upstream miR-125b-5p.2.qRT-PCR and double luciferase reporter gene experiments confirmed the targeted negative regulation ofmiR-125b-5p and EVA1 A.In D-Galn/LPS-induced L02 cell model,IL-1 β,TNF-α,Caspase-1 and NF-κ Bp65 were highly expressed,miR-125b-5p was significantly up-regulated and EVA1 A was significantly down-regulated.Changing the expression of miR-125b-5p affects the expression of EVA1 A and NF-κ Bp65,and then regulates the inflammatory level of L02 cells:up-regulation of miR-125b-5p can significantly inhibit the level of EVA1 A and increase the expression of NF-κ Bp65,while down-regulation of miR-125b-5p can increase the level of EVA1 A and inhibit the promoting effect ofNF-κBp65 on inflammation.3.The ALF mice model was successfully established and verified by hematoxylin-eo sin staining,ALT,AST and INR.The up-regulated expression of IL-1 β,TNF-α and Caspase-1 in liver tissue and serum was observed,and morphological changes,increased expression of inflammatory factors and pro-inflammatory mediators in liver tissue were observed by tail vein injection of agomiR-125b-5p and antagomiR-125b-5p.To verify that miR-125b-5p promotes the expression of NF--κBp65by targeting inhibition of EVA1 A,and then aggravates liver inflammation in mice.Part Ⅱ:1.The results of transmission electron microscope,nanoparticle tracking analysis and Western blot showed that KCs-Exo;could increase the secretion of KCs-Exo in both PBS and D-Galn/LPS,and there was significant difference between groups,and the KCs-Exo labeled with fluorescent dye could be absorbed by AM L12 cells and increased the level of mature miR-125b-5p in AML12 cells.AgomiR-125b-5p transfection or Exo-D-Galn/LPS treatment could lead to the activity of EVA1 A luciferase in AML12 cells,and the protein expression of EVA1 A was significantly decreased by Western blot detection.Transfection of agomiR-125b-5p or direct intake of KCs-Exo could promote the expression of GSDMD-N,indicating that scorch death increased,and both miR-125b-5p inhibitor and overexpression ofpcEVA1 A could inhibit the increase of GSDMD-N.2.In Exo-D-Galn/LPS group,the levels of serum ALT and AST were increased,and the concentrations of IL-1 β and Caspase-1 cytokines were increased;the results of Westernblot showed that the expression of GSDMD increased and the expression of EVA1 A decreased;the activation of GSDMD was inhibited after overexpression of EVA1 A,which could be blocked by Exo-D-Galn/LPS.3.The levels of IL-1 β,Caspase-1,GSDMD-N and EVAl A in patients with ALF were increased by Western blot,and the expression of specific proteins and miR-125b-5p in exocrine bodies of patients with ALF were significantly higher than those of normal subjects.Conclusion:1.In the process of ALF or inflammation,miR-125b-5p/EVA1 A signal axis can up-regulate the expression of NF-κB p65 and promote the release of inflammatory mediators,thus exacerbating the inflammatory injury of the liver.2.Exosomes derived from KCs can regulate the level of GSDMD through the miR-125b-5p/EVA1 A axis,promote the occurrence of hepatocyte scorch and accelerate hepatocyte death.更多还原