【作者】 刘晓斐；

【导师】 邹全明；

【作者基本信息】 第三军医大学 ， 临床检验诊断学， 2011， 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,已造成全世界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感染能够引起机体较强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,反而由于细菌的持续感染导致胃粘膜免疫病理损害。到目前为止,H. pylori慢性持续性感染及相关免疫机制并不清楚。细胞因子作为细胞间的信使分子,通过和靶细胞上的受体相结合,产生特定的生物学效应。H. pylori的慢性感染是细菌与宿主相互作用的结果,在此过程中有许多细胞因子参与并发挥了不同的功能。现已证明:CD4~+T细胞(Th细胞)在H. pylori感染过程中发挥重要作用。因此,目前关于H. pylori感染激发的细胞因子的研究多集中于Th细胞相关的细胞因子上。以往研究认为,IFN-γ主要参与宿主的炎症反应,并导致胃粘膜损伤甚至溃疡;而IL-4在一定程度上能够缓解胃炎,并降低H. pylori的定植。近年来研究发现一些新型Th细胞亚群如Th17,Th22,Th9等,这些细胞亚群在多种慢性炎症性疾病中发挥着重要作用。探明新型细胞亚群相关重要细胞因子的功能,将有助于增加以慢性炎症为机制相关疾病的理解,为慢性炎症疾病的免疫防治提供新的思路。IL-22是Th17及Th22细胞产生的重要效应因子,目前报道显示,IL-22通过诱导角质化细胞产生促炎症因子和趋化因子,介导炎症反应,在皮肤炎症性疾病中参与致病过程,而在肺炎克雷伯菌及啮齿枸椽酸杆菌感染模型中,IL-22通过调控局部炎症反应及刺激上皮细胞分泌抗菌肽等参与宿主防御反应。提示IL-22依据其所处的炎症微环境不同,具有发挥促炎致病或防御保护双重作用的特性。在我们的预实验中首次发现:H. pylori感染患者胃组织中IL-22呈异常高表达。那么在H. pylori引起的以慢性炎症为特征的感染性疾病中,IL-22发挥什么样的功能呢?目前尚未见报道。【研究目的】1.本课题拟以Th细胞重要效应因子IL-22作为主要研究对象,研究在H. pylori感染中的表达特性;2.初步探明IL-22在H. pylori感染中的作用及机制。【研究方法】1. IL-22在H. pylori感染中的表达特性研究收集消化内科胃炎患者胃粘膜组织,提取组织RNA,采用定量PCR检测细胞因子mRNA水平情况及H. pylori定植拷贝数;提取组织蛋白,ELISA检测IL-22蛋白表达;病理切片H&E染色检测胃组织炎症。部分患者采用耐信、阿莫西林克拉维酸钾和痢特灵三联疗法治疗,同时收集治疗后胃粘膜标本,定量PCR检测IL-22表达,病理切片H&E染色检测胃组织炎症,分析IL-22表达与H. pylori定植及胃组织炎症的相关性。2. IL-22~+ T细胞在H. pylori感染中的应答研究首先,建立H. pylori 26695感染细胞模型,定量PCR在细胞感染模型中检测分泌IL-22的细胞类型,并采用双重免疫荧光染色及流式在胃组织中进一步验证。随后,采用流式检测分泌IL-22的T细胞亚群在H. pylori感染患者胃组织中的应答情况。最后,采用定量PCR在CagA、UreB基因敲除株及出发菌株H. pylori 26695感染细胞模型及胃粘膜标本中鉴定诱导IL-22表达的H. pylori毒力因子。3. IL-22在H. pylori感染中的作用机制研究采用流式检测不同刺激因素下AGS细胞表面IL-22R1表达情况,定量PCR及组织免疫荧光染色检测胃组织中IL-22R1表达,明确H. pylori感染胃粘膜组织后IL-22R1的表达情况;IL-22刺激胃上皮细胞24h后,收集细胞,定量PCR检测促炎症因子及基质金属蛋白酶(MMP)表达,检测IL-22对胃上皮细胞的作用;采用定量PCR检测胃组织中促炎症因子及MMPs表达并分析与IL-22表达的相关性,初步探讨IL-22在H. pylori感染中的功能;体外采用Transwell细胞趋化实验检测IL-22对淋巴细胞的趋化作用,探讨IL-22促炎作用的机制。【研究结果】1. IL-22在H. pylori感染中的表达特性研究1.1 H. pylori阳性患者胃粘膜组织中,IL-22 mRNA水平及蛋白表达量显著增高(P < 0.05),同时检测结果表明,与H. pylori感染相关的细胞因子IL-17A,IL-17F和IFN-γmRNA水平亦显著增高(P < 0.05)。结果提示H. pylori感染后能够引起IL-22表达增加。1.2进一步分析发现胃粘膜组织IL-22 mRNA水平与H. pylori拷贝数呈显著正相关(r = 0.404, P < 0.01);根据病理切片H&E染色对胃炎严重程度进行分类统计,结果显示:中度和重度胃炎患者胃粘膜组织IL-22 mRNA水平显著高于轻度胃炎患者(P <0.05),而轻度胃炎患者胃粘膜组织IL-22 mRNA水平也显著高于正常胃粘膜组织(P < 0.05)。结果提示IL-22表达水平与胃粘膜炎症程度有关。1.3 H. pylori阳性患者经清除治疗后,胃粘膜组织IL-22 mRNA水平较治疗前显著降低(P < 0.05),并且H. pylori清除后胃组织炎症程度减轻(P < 0.05)。结果再次验证IL-22表达水平与H. pylori感染及炎症密切相关。2. IL-22~+ T细胞在H. pylori感染中的应答研究2.1 H. pylori 26695与AGS细胞共培养24h后,细胞形态呈明显“蜂鸟样”改变;细胞分泌大量促炎细胞因子IL-8,说明成功建立H. pylori感染细胞模型。H. pylori 26695感染胃上皮细胞株及T细胞株后检测到IL-22 mRNA水平显著增高(P < 0.05),同时胃组织双重免疫荧光染色IL-22蛋白及细胞表面标志发现,上皮细胞及CD4~+、CD8~+细胞均能够分泌IL-22。结果提示,H. pylori感染后胃上皮细胞和T细胞是胃组织分泌IL-22的细胞类型。2.2流式检测胃粘膜组织中分泌IL-22的细胞亚群类型:以CD3~+细胞圈门,CD4~+及CD8~+两个细胞亚群都能够分泌IL-22,且主要由记忆型T细胞分泌。进一步分析H. pylori感染阳性及阴性患者胃粘膜组织中细胞比率差异:以CD3~+细胞圈门,分别检测CD4~+及CD8~+ T细胞分泌细胞因子的比率。统计结果显示,H. pylori感染患者胃粘膜组织的IL-22~+ CD4~+, IL-22~+ CD8~+, IL-17~+ CD4~+, IL-17~+ CD8~+, IFN-γ~+ CD4~+细胞比率显著高于未感染者(P < 0.05),而IFN-γ~+ CD8~+细胞比率在两者间并无显著差异(P > 0.05);分别以CD4~+及CD8~+ T细胞圈门,H. pylori感染患者胃粘膜组织的IL-22~+ IL-17~+及IL-22~+ IFN-γ~+共表达细胞比率均显著高于未感染者(P < 0.05)。以上结果提示,IL-22~+ T淋巴细胞在H. pylori感染后应答增强。2.3分别采用CagA或UreB基因敲除株及出发菌株H. pylori 26695感染胃上皮细胞株及T细胞株,结果显示:CagA基因敲除株感染组相对于出发菌株H. pylori 26695感染组IL-22 mRNA水平明显降低(P < 0.05),而UreB基因敲除株组没有明显变化。随后,对胃组织标本进行CagA基因检测后发现,CagA基因阳性的H. pylori感染患者胃组织中IL-22 mRNA水平显著高于CagA基因阴性的H. pylori感染患者(P < 0.05),但IL-22mRNA水平在CagA基因阴性的H. pylori感染患者与无H. pylori感染者之间没有差异。结果提示,CagA是调控胃组织中IL-22表达的分子。3. IL-22在H. pylori感染中的作用机制研究3.1采用流式检测不同刺激因素下AGS细胞表面IL-22R1表达的结果显示,细胞因子及重组蛋白刺激AGS细胞后,IL-22R1表达没有明显变化,但H. pylori 26695菌株感染后,在一定感染范围内,IL-22R1表达量随细菌量增加而增加;胃组织免疫荧光染色显示胃组织中IL-22R1表达阳性,同时定量PCR检测结果显示,H. pylori阳性患者胃粘膜组织中IL-22R1 mRNA水平显著增高(P < 0.05),提示H. pylori感染后能够上调胃上皮细胞表达IL-22R1。3.2 IL-22刺激胃上皮细胞系24 h后,S100A8,S100A9,IL-8,MMP-1,MMP-10 mRNA水平均显著增高(P < 0.05);同时检测到H. pylori感染者胃组织中IL-8,S100A8,S100A9 mRNA水平亦显著增高(P < 0.05),而MMP-1,MMP-10 mRNA水平无明显变化;相关性分析显示胃粘膜组织中IL-22的表达与S100A8,S100A9,IL-8表达存在显著正相关(P < 0.05),而与MMP-1,MMP-10表达无明显相关性。结果提示,IL-22可能通过诱导上皮细胞产生促炎症因子等,参与炎症反应。3.3进一步采用Transwell细胞趋化实验检测IL-22对淋巴细胞的趋化作用,结果显示IL-22单独并不能趋化淋巴细胞,但与胃上皮细胞共培养24 h后能够趋化Transwell小室内的淋巴细胞(P < 0.05),而加入IL-8中和性抗体后,趋化的淋巴细胞数量显著降低(P < 0.05)。结果提示,IL-22通过诱导胃上皮细胞产生IL-8等趋化因子进而趋化炎症细胞,参与炎症反应。【结论】1. CagA~+H. pylori感染后能够诱导IL-22转录水平与蛋白表达上调,且IL-22表达与H. pylori定植拷贝数及粘膜炎症程度呈显著相关。2.胃组织中分泌IL-22的细胞主要为上皮细胞和记忆型T淋巴细胞,并且分泌IL-22的T淋巴细胞亚群在H. pylori感染患者的胃粘膜中应答增强;鉴定H. pylori毒力因子CagA是调控IL-22表达的分子。3.在H. pylori感染过程中,IL-22通过调控多种促炎症因子的表达,诱导炎症细胞浸润等参与炎症反应过程。【意义】深入研究H. pylori感染后IL-22的功能及其调控,将有助于我们更全面认识H. pylori感染的免疫应答规律及其致病机制,为H. pylori相关疾病的防治提供更充分的理论依据。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 persistent infection causes severe local inflammation in the gastric mucosa but the detailed pathogenic mechanism is not clear now.Cytokine as the message molecule induces specific biological effect. Interaction between pathogen and host results in H. pylori chronic infection and many cytokines play different roles during the infection. It’s proved that CD4~+ T cells (Th cells) play important roles in H. pylori persistent infection, so the study of H. pylori inducing cytokines focused on the Th cells produced. To date, it is generally accepted that gastric inflammation largely depends on IFN-γsecretion and IL-4 relieves inflammation and reduces H. pylori colonization.Recently, some novel subsets of effector T cell have been identified, such as Th17, Th22 and Th9. The recent studies have shown that novel T cell subsets have played important roles in inflammatory diseases. The study of cytokines produced by novel effector T cells may increase a better understanding of mechanism in chronic inflammation. The currently published data show that IL-22, which is produced by Th17 and Th22 cells, is an important mediator in dermal inflammation and it activates epithelial cells to secrete pro-inflammatory chemokines and cytokines. However, During K. pneumonia infection of the lung, C. rodentium infection of colon, IL-22 pathway have elucidated its essential protective role in mucosal immunity against these bacterial pathogens. It suggests that IL-22 is a dual-natured cytokine, depending on the context of inflammation, it can have either proinflammatory or protective properties. In our incipient study, we firstly found that IL-22 expression increased in H. pylori-infected gastric tissue. However, the characteristics and role of IL-22 in H. pylori infection have not been elucidated.【Objectives】 1. To characterize IL-22 expression in H. pylori infection.2. To elucidate the role of IL-22 in H. pylori infection and explore the mechanism.【Methods】1. The characteristic of IL-22 expression in H. pylori infected gastric tissueThe gastric biopsy specimens were collected in Gastroenterology department of XinQiao Hospital. Cytokines mRNA expression and the colonization of H. pylori were detected by real-time PCR and the protein level of IL-22 was examined by ELISA. The gastric inflammation was assayed by H&E staining. Some patients were treated with Nexium, Amoxicillin and Clavulanate Potassium and furazolidone for one week. The gastric biopsy specimens were collected after treatment and IL-22 expression was detected by real-time PCR and the gastric inflammation was assayed by H&E staining.2. The response of IL-22-producing T cell in H. pylori infected gastric tissueFirstly, the gastric epithelial cells model infected with H. pylori 26695 were established. IL-22 expression in H. pylori 26695 infected cell models was measured by real-time PCR and the major source of IL-22 was further detected by Double Immunofluorescence staining and FCM. Secondly, IL-22-producing T cell responses were examined by FCM in the gastric mucosa. Lastly, the virulence factor of H. pylori inducing IL-22 expression was identified by real-time PCR in H. pylori infected cell models and gastric tissue.3. The role of IL-22 in H. pylori infectionThe IL-22R1 expression was measured by FCM under the stimulation of various factors on AGS cell surface and real-time PCR and Immunofluorescence staining were used to detect the IL-22R1 expression in gastric tissue. Gastric epithelial cells were stimulated with commercial IL-22 for 24h and cells were collected for analysis of proinflammatory factors and MMPs mRNA expression by real-time PCR. Proinflammatory factors and MMPs mRNA expression were also detected by real-time PCR in gastric tissue and IL-22 expression was analyzed in relation to these factors expression. The influence of IL-22 on lymphocyte chemotaxis was detected by Transwell chemotaxis assay.【Results】1. The characteristic of IL-22 expression in H. pylori infected gastric tissue1.1 Gastric mucosal IL-22 mRNA and protein levels were significantly higher in H. pylori-positive patients than uninfected patients (P < 0.05), simultaneously, the IL-17A, IL-17F and IFN-γmRNA expression were also increased in H. pylori-infected patients (P < 0.05).1.2 Gastric mucosal IL-22 mRNA levels showed significant positive correlation with H. pylori copies in H. pylori-infected people (r = 0.404; P < 0.01). IL-22 mRNA expression in moderate and severe inflammation was higher than mild inflammation tissue and IL-22 mRNA in mild inflammation was also expressed at a high level than normal tissue (P < 0.05). These results showed that IL-22 expression was correlated with the degree of inflammation.1.3 IL-22 mRNA levels significantly decreased after a successful eradication treatment (P < 0.05) and H. pylori eradication results in a marked alleviation of gastric inflammation (P < 0.05). These results proved that IL-22 was correlated with H. pylori copies and inflammation.2. The response of IL-22-producing T cell in H. pylori infected gastric tissue2.1 After the incubation of H. pylori 26695 with the AGS cells for 24h, induction of the scattering phenotype and IL-8 release were observed. These results showed that we established a H. pylori 26695 infected cell model. IL-22 mRNA expression was significantly increased in the three gastric epithelial cell lines and T cell line by H. pylori 26695 infection (P < 0.05). IL-22 and cell marker protein expression was also detected in gastric tissue section by Double Immunofluorescence staining, suggesting that IL-22 was produced by epithelial cell and T cells.2.2 lymphocytes from gastric mucosa were isolated and detected by intracellular cytokine staining. The results showed that IL-22 was both produced by CD4 and CD8 T lymphocytes and the phenotype of IL-22-producing T cells displayed almost CD45RO positive, which showed that in the gastric tissue, the IL-22-producing T cells are mainly memory cells. Further, We found that the percentages of IL-22~+ CD4~+, IL-22~+ CD8~+, IL-17~+ CD4~+, IL-17~+ CD8~+, IFN-γ~+ CD4~+ T cells were significantly increased in H. pylori-infected patients as compared to H. pylori-negative individuals (P < 0.05), but no difference in IFN-γ~+ CD8~+ T cells. Lastly, we analyzed that percentages of IL-22~+IL-17~+ cells and IL-22~+IFN-γ~+ cells in the CD4 or CD8 gate were all increased in the H. pylori-infected patients (P < 0.05). These results showed that IL-22-producing T cells were increased in H. pylori-infected gastric mucosa.2.3 Three gastric epithelial cell lines and T cell line were infected with CagA mutant, UreB mutant or initial H. pylori 26695 strain for 24 h, IL-22 induction was reduced from gastric epithelial cells and T cells cocultured with CagA mutant ( P < 0.05 vs H. pylori 26695 strain), whereas IL-22 induction was similar to H. pylori 26695 strain using the UreB mutant. The IL-22 mRNA expression was also significantly higher in CagA positive patients (P < 0.05) and there was no difference between CagA negative and uninfected patients. These results suggested that CagA might be the virulence factor of H. pylori for regulating IL-22 expression.3. The role of IL-22 in H. pylori infection3.1 We detcted IL-22R1 expression on AGS cell surface under the stimulation of various factors by FCM and found that the mean fluorescence intensity (MFI) of IL-22R1 was upregulated with H. pylori 26695 infection, but no difference with cytokines and recombination proteins stimulation. Immunofluorescence staining showed that IL-22R1 expression was positive in gastric tissue and IL-22R1 mRNA expression in the gastric tissue was elevated in H. pylori-infected patients compared to the H. pylori-negative individuals (P < 0.05). These rusults indicated that H. pylori could regulate IL-22R1 expression in gastric epithelial cells.3.2 The gastric epithelial cells were stimulated with IL-22 for 24 h in vitro and the proinflammatory factors and MMPs expressions were detected by real-time PCR. The results showed that IL-22 significantly induced S100A8, S100A9, IL-8, MMP-1 and MMP-10 mRNA expressions in cell lines (P < 0.05). We also detected these gene expressions in the gastric tissue by real-time PCR. The results showed that IL-8, S100A8 and S100A9 were significantly increased in H. pylori-positive individuals (P < 0.05), but no differences in MMP-1 and MMP-10 expressions. We further analyzed the correlation between IL-22 and the proinflammatory factors expressions in the gastric tissue and found that IL-22 mRNA expression was significantly positive correlated with the IL-8, S1008A and S100A9 mRNA expressions, but not correlated with the MMP-1 and MMP-10 expressions. All these results suggested that IL-22 might participate the inflammatory reaction through producing pro-inflammatory factors in H. pylori infection3.3 The effect of IL-22 on lymphocytes was detected by Transwell chemotaxis assay. The results showed that there is no effect on lymphocytes with IL-22 alone, but after coculture with AGS cells for 24 h, IL-22 could induce much more lymphocytes in the lower chamber than AGS cells in chamber alone (P < 0.05). To determine whether this effect was mediated by IL-8, PBMCs were incubated with conditioned medium in the presence or the absence of neutralizing anti-IL-8 Ab. The anti-IL-8 Ab inhibited the chemotaxis of lymphocytes in response to IL-22 stimulated AGS cells (P < 0.05). These results suggested that IL-22 participated the inflammatory reaction by producing IL-8, which could recruit the lymphocytes in gastric tissue.【Conclusion】1. Gastric mucosal IL-22 expression was significantly higher in CagA positive H. pylori-infected patients than uninfected patients and IL-22 mRNA levels showed significant positive correlation with H. pylori colonization and inflammation in gastric tissues.2. The major source of IL-22 was epithelial cell and memory T cell and IL-22-producing T cells were significantly increased in H. pylori-infected patients as compared to H. pylori-negative individuals in the gastric mucosa. CagA was the virulence factor of H. pylori for regulating IL-22 expression.3. IL-22 participated inflammatory reaction by regulating proinflammatory factors expressions and inducing inflammatory cell infiltration during H. pylori infection.【Significance】Further study of the function and regulation of IL-22 after H. pylori infection may help to elucidate the characteristics of immune response and the pathogenic mechanism of H. pylori infection and to explore novel and effective therapies for H. pylori-associated diseases.更多还原