【作者】 石云；

【导师】 邹全明；

【作者基本信息】 第三军医大学 ， 生物技术， 2009， 博士

【摘要】 幽门螺杆菌(Helicobacter pylori,H. pylori)是一种螺旋状、微需氧革兰阴性杆菌,主要定植于人胃粘膜,已造成全球50%以上人口感染。H. pylori感染是慢性胃炎、消化性溃疡及胃粘膜相关淋巴组织淋巴瘤(MALT)等疾病的重要致病因子,与胃癌的发生密切相关,已被WHO列为Ⅰ类致癌因子。H. pylori感染可以引起机体较强烈的天然和获得性免疫应答,但自然感染H. pylori后的免疫反应并不能清除细菌,反而由于持续感染导致胃粘膜免疫病理损害。目前,H. pylori慢性持续性感染的机理仍不十分清楚。CD4+T细胞即Th细胞在H. pylori感染的免疫应答中发挥重要作用,Th细胞被激活后,通过不同的分化途径分化为效应性Th细胞,获得特定的生物学功能,根据其产生细胞因子和生物学功能的不同,传统上将效应性CD4+T细胞分为Th1和Th2亚群。H. pylori感染通常表现为粘膜组织内特异性CD4+T细胞增加,T细胞主要产生IL-2、IFN-γ、IL-12等细胞因子,提示为Thl型免疫应答。现多认为Thl型免疫应答参与了H. pylori感染慢性炎症的进程,并导致胃粘膜损伤甚至溃疡,但仅仅Th1型免疫应答并不能完全解释H. pylori感染后胃炎的发生。近年研究发现一类不同于Th1和Th2细胞的Th细胞亚群,此细胞亚群分泌IL-17、IL-6,IL-22和IL-21,而不分泌IL-4和IFN-γ,被命名为Th17细胞亚群。Thl7细胞主要介导慢性炎症,在自身免疫病、炎症性疾病及防御粘膜胞外菌感染中发挥重要作用。Thl7细胞的发现及其分化和功能的研究,有助于我们对以慢性炎症为机制的疾病的理解和阐明,为慢性炎症疾病的免疫防治提供了新的思路。那么Th17细胞在H. pylori感染过程中呈现什么样的应答规律,在H. pylori感染中发挥什么样的功能呢?目前还不清楚。【研究目的】1.本研究拟以Th17细胞及其主要效应因子IL-17作为研究的主要对象,研究其在H. pylori感染中的应答规律;2.初步明确Th17细胞在H. pylori慢性感染中的作用及初步机制探讨。【研究方法】1.建立H. pylori感染BALB/c小鼠模型,在H. pylori感染后不同时间点处死小鼠,取胃组织提取RNA检测细胞因子mRNA水平变化,提取胃组织蛋白,ELISA检测细胞因子蛋白水平变化,取小鼠的脾,肠系膜淋巴结,胃旁淋巴结分离淋巴细胞后,细胞内因子染色,FCM检测Th1,Th2和Th17细胞应答。2.基因工程方法制备GST-IL-17融合蛋白,免疫家兔制备抗IL-17抗体,鉴定其中和IL-17的活性。口服灌胃H. pylori前腹腔注射抗IL-17抗体,real-time PCR检测阻断IL-17效应后H. pylori定植量的变化,H&E染色检测胃组织炎症;并采用AdmIL-17口服灌胃后感染H. pylori,检测调控IL-17高表达对H. pylori定植量及胃组织炎症的影响。3.培养小鼠胃上皮细胞(MFC),加入IL-17刺激24 h,收集细胞,抽提RNA,real-time PCR检测基质金属蛋白酶(MMP )和趋化因子的变化,并在上述IL-17阻断和高表达模型中检测MMP和趋化因子的变化。【研究结果】1. Th17细胞及相关细胞因子在H. pylori感染中的应答规律1.1 H. pylori感染组的小鼠胃组织中IFN-γ和IL-17的表达在mRNA及蛋白水平均显著增高,而IL-4的表达在蛋白水平没有显著的变化。调控Th1和Th17细胞应答的细胞因子IL-12和IL-23的表达在H. pylori感染后也有显著升高。1.2通过流式细胞技术检测Th细胞的频率发现:H. pylori感染小鼠的脾淋巴细胞中H. pylori特异性的Th1和Th17细胞的比率显著高于正常小鼠的脾淋巴细胞,而Th2细胞应答变化不显著。提示H. pylori感染可以诱导特异性的Th1和Th17细胞应答。1.3 Th细胞应答呈现动态变化:H. pylori感染后7 d,在肠系膜淋巴结检测到Th1,Th2及Th17细胞的应答,在脾淋巴细胞中,Th17细胞在14 d开始增多,21 d达到高峰,Th1细胞在21 d开始增多,而Th2细胞在整个感染进程中没有显著变化。在胃旁淋巴结中Th细胞的应答较晚在28 d出现应答,其Th1及Th17细胞应答的规律与脾的情况类似。脾和胃旁淋巴结的淋巴细胞中Th细胞应答显示Th17细胞应答要早于Th1细胞应答,并且不同淋巴器官中Th细胞应答的时间提示肠系膜淋巴结可能是H. pylori激发特异性免疫应答较早的场所。2. Th17/IL-17促进H. pylori的定植以及胃组织炎症2.1兔抗IL-17抗体处理组的小鼠H. pylori定植量及胃组织炎症显著均显著低于对照IgG处理组小鼠(P < 0.05)。结果提示中和IL-17的活性有助于H. pylori的清除并减轻炎症,说明Th17/IL-17途径可能削弱了抗H. pylori感染的宿主防御。2.2我们采用AdmIL-17感染胃组织上调IL-17的表达后再感染H. pylori,检测H. pylori定植量和胃组织炎症的变化。AdmIL-17处理小鼠,与AdLuc和PBS处理组相比,在H. pylori感染后显著增加H. pylori的定植量(P < 0.01),而AdLuc处理组与PBS处理组相比,H. pylori的定植量没有显著变化(P > 0.05)。H&E染色切片显示,AdmIL-17组小鼠胃组织炎症程度强于AdLuc处理组(P < 0.0125),而AdLuc处理组小鼠的胃粘膜炎症与PBS组相比没有显著性变化(P > 0.05)。这些结果显示Th17 / IL-17可能增加了宿主对H. pylori感染的敏感性。3. Th17细胞/IL-17在H. pylori感染中的作用机制初探3.1小鼠胃上皮细胞系MFC细胞在IL-17的刺激下,MMP-2,MMP-3,MMP-7,MMP-9和趋化因子CCL2,CCL5,CCL20,CCL25以及CXCL1等mRNA表达与未刺激的细胞相比均显著增高(P < 0.05)。提示MMP和趋化因子可能参与了H. pylori感染中IL-17介导的免疫效应。3.2 IL-17抗体处理后H. pylori感染胃组织中MMP-9和CCL25的表达与对照IgG处理小鼠相比显著降低(P < 0.05),而其他MMP和趋化因子的表达没有显著变化。3.3 AdmIL-17处理后H. pylori感染组,与AdLuc和PBS处理组相比,MMP-2, MMP-9和CCL25的表达显著增高(P < 0.05)。以上结果提示我们Th17细胞及IL-17可能通过诱导MMP-9以及CCL25的表达参与调控H. pylori感染及炎症反应。【主要结论】1. H. pylori感染后可以诱导H. pylori特异性的Th1和Th17细胞应答。Th17细胞应答要早于Th1细胞应答,并且不同淋巴器官中Th细胞应答的时间提示肠系膜淋巴结可能是H. pylori激发特异性免疫应答较早的场所。2. Th17/IL-17可以促进H. pylori感染后的细菌定植以及炎症的程度。3. Th17/IL-17可能是通过调控MMP以及趋化因子的表达参与H. pylori感染后的细菌定植以及炎症的调控。【研究意义】Th17/IL-17可能成为H. pylori感染相关疾病辅助治疗的靶标,深入了解H. pylori感染后Th细胞免疫应答特征,功能及其调控,将有助于我们更全面了解H. pylori感染的致病机制,并且有助于指导H. pylori相关免疫疾病的免疫治疗。更多还原

【Abstract】 Helicobacter pylori (H. pylori) is a Gram-negative, microaerophilic bacterium that resides extracellularly in the gastric mucosa and infects more than 50% of the population worldwide. H. pylori-induced chronic inflammation is the cause of gastritis and peptic ulcer and a risk factor for gastric cancer. H. pylori infection causes severe local inflammation in the gastric mucosa.CD3+ CD4+ T cells are increased in infected gastric lamina propria and play important roles in the pathogenesis of persistent H. pylori infection. Traditionally, CD4+ T cells, also known as T helper cells (Th cells), are classified into two main classes: Th1 and Th2, on the basis of their cytokine secretion and immune regulatory functions. Th1 cells secrete IFN-γ, IL-2, and IL-12 and regulate cellular immunity, while Th2 cells produce IL-4, IL-5, and IL-13 and mediate humoral responses. To date, studies of immune responses to H. pylori have largely focused on Th1 and Th2 cells and it is generally accepted that H. pylori infection results in a Th1-dominant response and that gastric inflammation largely depends on Th1 cell responses, however, IFN-γsecretion alone is insufficient to induce gastritis. Thus, the detailed mechanism of pathogenesis is not clear.Recently, a novel subset of effector T cells, identified by secretion of IL-17, has been defined as Th17 cells. Th17 cells are distinct from Th1 and Th2 cells in differentiation and function. Th17 cells are clearly implicated in the pathogenesis of autoimmune diseases by promoting chronic inflammation. However, the protective and pathogenic functions of IL-17-producing Th cells were both reported in infections. The identification of Th17 cells necessitates a re-evaluation of Th cell responses in H. pylori infection. However, the characteristics of Th cell responses, including those of Th1, Th2 and Th17 cells, remain unclear and the role of Th17 cell responses in H. pylori infection has not been elucidated.【Objectives】1. Try to characterize Th cell responses, especially Th17 cell responses, to H. pylori shortly after infection of a BALB/c mouse model.2. Aim to elucidate the role of Th17 cells in the early stage of H. pylori infection and explore the mechanism.【Methods】1. BALB/c mice were infected with H. pylori orally and gastric tissues were collected at the indicated times post-infection. Cytokine expression in the stomach was determined by real-time PCR and ELISA. Cell responses in spleen,mesenteric lymph node and paragastric lymph node were examined by intracellular staining for IFN-γ, IL-4 and IL-17.2. The recombinant GST-IL-17 was cloned and expressed in E. coli and were used to immunize rabbit to obtain the anti-IL-17 antibody. Before H. pylori infection, IL-17 antibody was administrated to the mice intraperitoneally and the colonization of H. pylori was measured with real-time PCR to detect the 16s rDNA of H. pylori and the gastric inflammation was assayed by H&E staining. In addition, AdmIL-17 was administrated to the mice orally before H. pylori infection to assay the effects of IL-17 on the colonization and inflammation.3. MFC cells were stimulated with commercial mIL-17, Cells were collected for analysis of MMP and chemokine mRNA expression by real-time PCR. The expression of MMP and chemokine mRNA was also detected in the IL-17 Ab or AdmIL-17 treated and H. pylori infected mouse model.【Results】1. H. pylori induced specific Th1 /Th17 cell responses1.1 Both mRNA and protein of IFN-γand IL-17 were expressed at a higher level in gastric tissue from H. pylori-infected mice than in those from control mice, whereas IL-4 expression did not change significantly in protein level. IL-23 and IL-12 were also increased in gastric tissue.1.2 Splenic lymphocytes from infected mice showed significantly higher Th1 and Th17 cell responses than those from uninfected mice when stimulated with PMA and ionomycin or H. pylori whole cell protein, whereas Th2 cell responses were not significantly changed, suggesting that specific Th1 and Th17 cell responses were induced in H. pylori infection. 1.3 Th1, Th2 and Th17 cell responses in MLN were all primed on day 7 pi. In the spleen, Th17 cells were expanded on day 14 and peaked on day 21 pi, and Th1 cells were induced from day 21 to 28 pi, whereas Th2 showed no significant change during the course of infection. The Th cell responses were delayed in PLN, reaching detectable levels on day 28 pi. The Th cell responses in spleen and PLN showed a dynamic change: Th17 cell responses preceded Th1 cell responses. The time course of Th cell responses in different organs suggests that MLN may be an earlier site of T cell priming during H. pylori infection.2. Th17/IL-17 contribute to gastric inflammation and H. pylori colonization.2.1 The colonization of H. pylori and the inflammation of gastric tissue in the anti-IL-17 Ab-treated mice was significantly lower than that in the control IgG-treated mice (P < 0.05),suggesting that the Th17/IL-17 pathway might impair host defense against H. pylori infection.2.2 Pretreatment with AdmIL-17 resulted in more copies of H. pylori in the stomach than in mice treated with AdLuc (P < 0.01), while the copies of H. pylori after AdLuc pretreatment were not different from those in PBS-treated mice (P > 0.05). In addition, mice treated with AdmIL-17 showed significantly more gastric inflammation than mice treated with AdLuc (P < 0.0125), and the inflammatory response in AdLuc-treated mice was similar to that in PBS-treated mice (P > 0.05). These results indicate that Th17/IL-17 might increase the susceptibility to H. pylori infection.3. The effect of IL-17 on expression of MMPs and chemokines3.1 The expression of MMP-2, MMP-3, MMP-7, MMP-9 and chemokine CCL2, CCL5, CCL20, CCL25 and CXCL1 in MFC cells were up-regulated by IL-17 stimulation.3.2 IL-17 Ab pretreatment reduced MMP-9 and CCL25 expression in H. pylori-infected stomachs (P < 0.05), but it had no significant effect on the expression of other factors.3.3 AdmIL-17 treatment increased MMP-2, MMP-9 and CCL25 expression compared with AdLuc-treated and PBS-treated mice (P < 0.05). The results indicate that Th17/IL-17 pathway may exert its effect on H. pylori infection by enhancing MMP-9 and CCL25 production.【Conclusion】 1. H. pylori-specific Th1/Th17 responses are induced in the BALB/c mouse model and display different dynamics. The Th cell responses occurred earlier in the mesenteric lymph node than in the spleen or paragastric lymph nodes.2. Th17/IL-17 pathway might facilitate bacterial colonization and inflammation in the early stage of H. pylori infection.3. The role of IL-17 on H. pylori infection might be associated with the expression of MMP-9 and CCL25.【significance】The Th17/IL-17 pathway might be a potential therapeutic target in H. pylori-associated diseases. Further study is required to elucidate the mechanisms by which H. pylori interacts with Th17 cells and how these cells facilitate infection. A better understanding of the nature, regulation, and function of Th cell responses to H. pylori may help to explore novel and effective immunotherapies for gastric diseases induced by this organism.更多还原