【作者】 郑伟；

【导师】 江米足；

【作者基本信息】 浙江大学 ， 临床医学（专业学位）， 2019， 博士

【摘要】 幽门螺杆菌(Helicobacter pylori,HP)感染是儿童慢性胃炎和消化性溃疡的主要病因,与胃粘膜相关淋巴组织淋巴瘤和胃癌的发病机制密切相关。一般认为,HP是细胞外病原体,但越来越多的证据表明该细菌可以在胃上皮细胞中增殖,免疫反应很难去除细胞内的HP并引起胃部疾病的发生。正常平衡的微生物菌群对于建立和维持宿主免疫平衡中起着至关重要的作用。越来越多的动物和临床研究表明,胃肠道微生物群可以显著影响疾病免疫治疗的临床效果。HP感染能够引起机体天然和获得性免疫应答,可诱发细胞免疫和体液免疫反应。目前关于HP感染相关的细胞因子研究多集中于Th细胞相关的细胞因子。根据不同的细胞因子谱和生物学功能,效应性T细胞分为辅助性T细胞(Th1、Th2)和调节性T细胞(Treg)亚群。Treg是具有调节功能的T细胞亚群,其在免疫抑制作用中不同于Th1和Th2效应性T细胞,并且在多种免疫性疾病中发挥重要的调节作用,近年来已成为免疫学领域的热点。根据其表面标志物、产生的细胞因子和作用机制,Treg可分为CD4+CD25+Treg、Treg1和Th3等[4,5]。胃微生物群是胃黏膜免疫必不可少的调节因子。共生菌群的特定成员,如梭菌属和脆弱拟杆菌(荚膜多糖A产生),是天然forkhead盒蛋白3(Foxp3+Tregs)的强诱导剂。Foxp3+Tregs表达CD4+CD25+Treg,在调节胃肠道免疫稳态、促进外周耐受性的建立和维持方面的重要性在动物和人类实验中都得到了证实[6-8]。既往研究发现,寡养单胞菌具有免疫刺激作用,可诱导TNF-α表达,对炎症反应有明显的促进作用。进一步研究发现许多不占优势的胃细菌与两种主要的免疫抑制细胞有显著的相关性。这些细菌大多属于变形杆菌门和厚壁菌门,它们单独或与细菌代谢产物一起调节胃黏膜免疫[9]。既往研究大多以肠道环境为基础,侧重于微生物群与黏膜免疫之间的相互作用,而这些相互作用在胃中的研究相对较少。HP感染引起Tregs及Th17的活化和细胞因子的改变,可能引起胃炎和消化性溃疡。目前尚不清楚HP感染的胃黏膜相关微生物群与Tregs的改变是否相关及如何相关。HP感染在引起机体免疫反应的同时,还可诱导胃上皮细胞产生自噬,通过自噬可降解病原体而起到保护细胞的作用。研究发现HP诱导的自噬通过降解胞内的VacA减少了 HP对胃上皮细胞造成的损伤;但同时又观察到在自噬体中的HP只有一部分得到了清除,而另一部分HP能在自噬体中生存、增殖。目前HP激活宿主的自噬过程仍未完全明确,尽管涉及不同的细菌和宿主因素,但使用不同的菌株和宿主细胞系会产生不同的结果。HP如何调控或阻断自噬介导的病原体向溶酶体的转运有利于其自身在自噬体中的存活和复制的呢?研究发现除HP外,其它多种细菌均可操纵自噬(如沙门氏菌、志贺菌等),可以调节细胞膜表面,也可以表达效应器和毒素,干扰、操纵自噬,增加细胞内复制和进一步入侵的潜力。那么HP感染是否影响改变了胃黏膜相关微生物菌群呢?成人的研究报道了HP感染者胃黏膜菌群以HP为主,同时伴随着其他菌属的相对耗竭。HP感染患儿胃黏膜微生物群落的研究很少,国外研究提出HP感染患儿胃黏膜菌群中HP占绝对优势,但涉及样本量少。中国儿童HP感染状态下胃黏膜菌群结构特征及其对黏膜免疫功能的影响尚未见报道。综合上述,我们做出以下推测:HP感染影响改变了胃黏膜相关微生物菌群,改变的微生物菌群影响Tregs的合成与分泌,进而影响胃黏膜局部免疫功能和炎症反应。HP感染影响改变了胃黏膜相关微生物菌群,改变的微生物菌群是否影响胃上皮细胞的自噬过程呢?因此,研究微生物菌群对自噬调节有可能揭示HP慢性持续性感染引起的疾病发生机制。因此本研究拟结合动物实验,探讨HP感染胃黏膜相关微生物菌群改变与黏膜免疫因子、黏膜细胞自噬及相关通路的关系,证明HP感染对胃黏膜相关微生物菌群的影响及其调节局部免疫功能的作用及机制。目的:1.了解HP感染对胃黏膜及十二指肠黏膜相关微生物菌群的影响。2.明确HP感染对胃黏膜组织免疫相关因子及细胞因子水平的影响。3.阐明HP感染胃黏膜诱导自噬发生的过程及其信号通路变化。方法:选择从2016年12月至2017年8月因“反复腹痛、腹胀、恶心、呕吐”等症状就诊于浙江大学医学院附属儿童医院的患儿122例,分为三组:HP感染胃炎组患儿(HP 阳性组)57例,HP阴性胃炎组患儿(HP阴性组)37例,HP阴性胃黏膜镜下及组织病理学均无异常的患儿(对照组)28例,分别活检胃窦部和十二指肠球部黏膜进行以下研究。1.HP感染对儿童胃黏膜以及十二指肠黏膜微生态的影响取每例患儿胃窦部及十二指肠球部黏膜各1块,提取黏膜总DNA,通过PCR提取16S rDNA V4区序列,利用Illumina Miseq高通量测序平台测序测定三组间胃黏膜及十二指肠黏膜相关微生物菌群多样性变化。2.HP感染对胃黏膜组织免疫相关因子及细胞因子水平的影响及机制对于HP阳性组、HP阴性组和对照组儿童胃镜下活检的胃窦部黏膜,每组随机选取12例,提取胃窦部黏膜总RNA。采用实时定量聚合酶链反应分析FOXP3、IL-10、TGF-β、TNF-α、IFN-γ、IL-1β、IL-6、IL-8、IL-12、IL-17A、IL-21、IL-23的基因表达情况。免疫组织化学染色观察CD4+T细胞及巨噬细胞(CD68)的表达情况。分析HP感染患儿CD4+T细胞、CD68细胞、FOXP3、IL-10、TGF-β、IL-17A、IL-23的表达特点及其相互关系。3.HP感染胃黏膜组织自噬发生过程及其信号通路变化每例患儿胃窦部黏膜各1块,分别用于提取组织总蛋白。另有6例(每组2例)活检胃窦部黏膜各1块予2.5%戊二醛固定后进行透射电镜观察,检测HP感染患儿胃黏膜组织细胞自噬小体形成。应用免疫印迹(Western-blot)技术检测HP感染患儿胃黏膜标本自噬标志性蛋白、自噬相关蛋白及相关信号通路蛋白。结果:1.基于细菌高通量测序分析证实儿童胃及十二指肠黏膜均存在大量细菌群落,且具有其独特的菌群特征和多样性。HP感染显著影响胃黏膜相关微生物群的α多样性和β多样性,但十二指肠黏膜相关微生物群落多样性影响不大。HP 阳性组患儿和HP阴性组患儿,组间胃黏膜细菌群落结构和多样性均存在显著差异,和HP阴性组相比,HP阳性组的6个门、5个纲、9个目、10个科、8个属的丰度水平显著降低;只有螺杆菌属的比例更为丰富。HP 阳性组患儿、HP阴性组患儿和对照组儿童十二指肠黏膜细菌群落结构和多样性均无显著差异。HP感染对十二指肠黏膜相关微生物群影响不明显,组间多个分类水平丰度无显著性差异。2.HP 阳性组 FOXP3、TGF-β1、IL-10、IL-17A、IL-23 的 mRNA 表达明显高于HP阴性组及对照组(p<0.01)。HP 阳性组FOXP3 mRNA水平与TGF-β1、IL-10的 mRNA 水平正相关(r=0.836,p<0.01;r=0.711,p<0.01),FOXP3 mRNA 水平与HP丰度正相关(r=0.833,p<0.001)。HP 阳性组CD4+T细胞及CD68细胞的数量均明显高于HP阴性组和对照组(p<0.05),CD4+T细胞的数量与炎症评分正相关(r=0.795,p<0.001),IL-17A mRNA/FOXP3 mRNA 比率与 HP 丰度负相关(r=-0.717,p<0.01)。HP 阳性组 IL-1β、IL-6、IL-8、IL-12、TNF-α、IFN-y、IL-21的表达与HP阴性组和对照组相比无统计学差异(p>0.05)。3.Westem-blot检测显示HP 阳性组较HP阴性组及对照组自噬标志性蛋白LC3表达水平明显降低,而P62表达水平明显增高;自噬相关蛋白Beclin-1、Atg5、Atg16L1表达量较HP阴性组及对照组均有不同程度的降低;磷酸化Akt蛋白水平较HP阴性组及对照组降低,而磷酸化的mTOR水平增高。结论:1.HP感染显著影响胃黏膜微生物群,降低了胃黏膜微生物群多种分类水平的丰度。与HP阴性组相比,HP感染对胃黏膜微生物群的影响更大。2.IL-17A/FOXP3对调节性T细胞偏向反应的平衡有利于HP持续感染,导致慢性活动性胃炎。3.HP感染可能是通过PI3K/Akt/mTOR信号途径参与了对自噬的抑制作用。更多还原

【Abstract】 Helicobacter pylori(H.pylori)infection is the main pathogenic factor of chronic gastric and peptic ulcer,which is closely related to the incidence of gastric mucosa-associated lymphoma and gastric cancer.It was generally believed that H.pylori was an extracellular pathogen,but more and more evidence showed that this bacterium can survived and proliferate in gastric epithelial cells,and the immune response of the body can clear the intracellular bacterium,leading to the occurrence of stomach diseases caused by persistent infection.Normally balanced microbial flora plays a vital role in establishing and maintaining the host immune balance.More and more animal and clinical studies show that gastrointestinal microbiota can significantly affect the clinical effect of immunotherapy for diseases.H.pylori infection can induce the body’s natural and acquired immune response and induce cellular and humoral immune responses.At present,the researches on the cytokines related to H.pylori infection mainly focus on the cytokines related to Th cells.According to different cytokine profiles and biological functions,T cells are dividedinto helper T cells(Th1,Th2)and regulatory T cells(Treg)subsets.Treg is a T cell subgroup with regulatory function,which is different from Th1 and Th2 effector T cells in immunosuppression,and plays an important regulatory role in a variety of immune diseases.In recent years,Treg has become a hotspot in the field of immunology.According to its surface markers,cytokines produced and mechanism of action,Treg can be divided into CD4+CD25+Treg,Treg1 and Th3.Gastric microbiota is an essential regulatory factor for gastric mucosal immunity.Specific members of the symbiotic flora,such as clostridium and bacteroides fragilis(pod polysaccharide A production),are strong inducers of natural forkhead box protein 3(Foxp3+Tregs).Foxp3+Tregs expressed CD4+CD25+Tregs,and its importance in regulating gastrointestinal immune homeostasis and promoting the establishment and maintenance of peripheral tolerance has been confirmed in both animal and human experiments.Previous studies have found that oligomonas has an immune stimulation effect,which can induce the expression of TNF-a and significantly promote the inflammatory response.Further studies showed that many of the less dominant gastric bacteria were significantly correlated with two major immunosuppressive cells.Most of these bacteria belong to proteobacteria and firmicutes,which regulate gastric mucosal immunity alone or together with bacterial metabolites.Most previous studies have been based on the intestinal environment,focusing on the interaction between microbiota and mucosal immunity,while relatively few studies have been conducted on these interactions in the stomach.H.pylori infection causes activation of Tregs and Th17 and changes in cytokines,which may cause gastritis and peptic ulcer.At present,it is not clear whether and how the gastric mucosa-related microbiota with H.pylori infection is related to the changes in Tregs.H.pylori infection can induce autophagy in gastric epithelial cells,which can degrade pathogens and protect cells.It was found that H.pylori-induced autophagy reduced the damage of gastric epithelial cells caused by H.pylori by degrading intracellular VacA.At the same time,it was observed that only part of H.pylori in autophagosome was cleared,while the other part could survive and proliferate in autophagosome.Currently,the autophagy process of H.pylori-activated host is still not completely clear.Although different bacterial and host factors are involved,different strains and host cell lines can produce different results.How does H.pylori regulate or block autophagy-mediated transport of pathogens to lysosomes in favor of its own survival and replication in autophagosomes?Studies have found that in addition to H.pylori,a variety of other bacteria can manipulate autophagy(such as salmonella,shigella,etc.),can regulate the surface of the cell membrane,and can also express effector and toxin,interfere with and manipulate autophagy,and increase the potential for intracellular replication and further invasion.So does H.pylori infection affect and alter the gastric mucosal microbiota?The study of adults reported that the gastric mucosal flora of H.pylori-infected patients was dominated by H.pylori,accompanied by relative depletion of other bacteria.There are few studies on the gastric mucosal microbial community of children infected with H.pylori.Foreign studies suggest that H.pylori occupies an absolute advantage in the gastric mucosal microbial community of children infected with H.pylori,but the sample size is small.The structural characteristics of gastric mucosal microbiota and its effect on mucosal immune function in Chinese children with H.pylori infection have not been reported.Based on the above,we made the following speculation:H.pylori infection affected the changes in the microbiota related to gastric mucosa,and the changes in the microbiota affected the synthesis and secretion of Tregs,thereby affecting the local immune function and inflammatory response of gastric mucosa.H.pylori infection affects the microbial community related to gastric mucosa.Does the altered microbial community affect the autophagy process of gastric epithelial cells?Therefore,the study on the regulation of autophagy by microbiota may reveal the pathogenesis of H.pylori chronic persistent infection.Therefore,this study intends to explore the relationship between changes in gastric mucosa-related microbiota of H.pylori infection and mucosal immune factors,mucosal autophagy and related pathways in combination with animal experiments,so as to prove the influence of H.pylori infection on gastric mucosa-related microbiota and the role and mechanism of regulating local immune function.Objectives:1.Investigate the changes of microbiota related to gastric mucosa and duodenal mucosa with H.pylori infection.2.Determine the effects of H.pylori infection on immune-related factors and cytokines in gastric mucosa.3.Elucidate the process and signal pathway changes of autophagy induced by H.pylori infection in gastric mucosa.Methods:From December 2016 to August 2017,122 children who were admitted to the children’s hospital,Zhejiang university school of medicine due to symptoms of"repeated abdominal pain,abdominal distension,dyspepsia,nausea and vomiting" were selected.There were three groups:57 children in the HP infection group(HP positive group),37 children in the HP negative gastritis group(HP negative group),and 28 children in the HP negative normal mucosa group(control group).Four gastric antrum mucosa biopsy samples and two duodenal bulb mucosa samples were collected for the following experiments.1.Influence of H.pylori infection on microbiota of gastric mucosa and duodenal mucosa in children.Total mucosal DNA was extracted from 1 piece of each group’s gastric antrum and duodenal ball mucosa,and 16S rDNA V4 region sequence was extracted by PCR.The diversity of microbiota related to gastric mucosa and duodenal mucosa in the three groups was analyzed by Illumina Miseq high-throughput sequencing platform.2.Study on immune regulation and mechanism of gastric mucosa in children induced by H.pylori infection.For the gastric antrum mucosa of the children in the HP positive group,the HP negative group and the control group,12 cases were randomly selected from each group.One piece of gastric antrum mucosa was taken from each group,and total RNA was extracted.The gene expression of FOXP3,IL-10,TGF-β,TNF-α,IFN-y,IL-1,IL-6,IL-8,IL-12,IL-17A,IL-21 and IL-23 was analyzed by real-time quantitative polymerase chain reaction.The expression of CD4+T cells and macrophages(CD68)was observed by immunohistochemical staining.The expression characteristics and correlation of CD4+T cells,CD68 cells,FOXP3,IL-10,TGF-β,IL-17A and IL-23 in children with HP infection were investigated.3.Study on the mechanism of autophagy regulation and persistent infection in gastric mucosa of children induced by H.pylori infection.One piece of each gastric antrum mucosa was taken from each group and used to extract total protein,and another 6 cases(2 cases in each group)were observed by 2.5%glutaraldehyde for fixed transmission electron microscope observation.Western blot was used to detect autophagy signature proteins,autophagy related proteins and related signaling pathways in gastric mucosa specimens infected with H.pylori,and transmission electron microscopy was used to detect autophagy vesicle formation in gastric mucosa specimens infected with H.pylori.Results:1.High-throughput bacterial sequencing analysis confirmed the existence of a large number of bacterial communities in the gastric and duodenal mucosa of children,with unique bacterial community characteristics and diversity.The presence of HP significantly affected the Alpha diversity and Beta diversity of gastric mucosa related microbioys,but not the diversity of duodenal mucosa related microbiota.There were significant differences in the structure and diversity of gastric mucosal bacterial community between HP positive group and HP negative gastritis group.The abundance level of 6 phyla,5 classes,9 orders,10 families and 8 genera in the HP positive group was significantly lower than that in the HP negative group,Only the proportion of helicobacter is more abundant.There was no significant difference in the structure and diversity of the duodenal mucosal bacterial community between HP positive group,HP negative gastritis group and HP negative mucosa normal group.There was no significant difference in the structure and diversity of the duodenal mucosal bacterial community between HP-positive group,HP-negative group and the control group.The presence of HP had no significant effect on the duodenal mucosal microbiota,and there was no significant difference in the abundance of multiple classification levels between the groups.2.The mRNA levels of FOXP3,TGF-1,IL-10,IL-17A and IL-23 in HP positive group were significantly higher than those in HP negative group and the Control group(p<0.01).In HP-positive group,the FOXP3 mRNA level was positively correlated with the mRNA level of TGF-1 and IL-10(r=0.836,p<0.01;r=0.711,p<0.01),and the FOXP3 mRNA level was positively correlated with the abundance of HP(r=0.833,p<0.001).The number of CD4+T cells and CD68 cells in HP positive group was significantly higher than that in HP negative group and the Control group(p<0.05),and the number of CD4+T cells was positively correlated with the inflammatory score(r=0.795,p<0.001).The mRNA ratio of IL-17A/FOXP3 was negatively correlated with HP abundance(r=-0.717,p<0.01).The expressions of IL-1,IL-6,IL-8,IL-12,TNF-α,IFN-γ,and IL-21 in HP positive group were not significantly different from those in HP negative group and the Control group(p>0.05).3.Western-blot analysis showed that the expression level of autophagy marker protein LC3 in HP positive group was significantly lower than that in HP negative group and the Control group,while the expression level of P62 was significantly higher.Beclin-1,Atg5 and Atg16L1 expression levels of autophagocyte-related proteins were significantly lower than those of the HP negative group and the Control group.The level of phosphorylated Akt protein was lower than that of HP negative group and Control group,while the level of phosphorylated mTOR was higher.Conclusions:1.The presence of HP significantly affected the gastric mucosa related microbiota,resulting in low abundance of multiple classification levels.Compared with HP negative group,HP infection had a greater effect on gastric microflora.2.The balance of IL-17A/FOXP3 in response to regulatory T cell bias favors the persistence of bacteria,leading to chronic active gastritis.3.HP infection may be involved in the inhibition of autophagy through the PI3K/Akt/mTOR signaling pathway.更多还原