IL-22在幽门螺杆菌感染中的表达特性及作用机制研究
发布时间：2018-08-12 07:57
【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HE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HE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HE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。
[Abstract]:Helicobacter pylori (H. pylori) is a spiral, microaerobic gram-negative bacilli, mainly colonized in human gastric mucosa, has caused infection in more than 50% of the world's population. H. pylori infection is an important pathogenic factor of chronic gastritis, peptic ulcer and gastric mucosa-associated lymphoid tissue lymphoma (MALT), and gastric cancer. H. pylori infection can induce strong natural and acquired immune response, but the immune response after natural infection can not eliminate bacteria, but because of the persistent infection of bacteria, gastric mucosal immune pathological damage. So far, H. pylori chronic persistent sexuality. The mechanism of infection and related immunity is not clear.
Cytokines, as messenger molecules between cells, produce specific biological effects by binding to receptors on target cells. Chronic infection of H.pylori is the result of interaction between bacteria and host. Many cytokines participate in and play different roles in this process. It has been proved that CD4~+T cells (Th cells) are sensitive to H.pylori. Therefore, the current research on cytokines stimulated by H. pylori infection is mostly focused on Th cell-related cytokines. Previous studies have shown that IFN-gamma is mainly involved in the host inflammatory response, leading to gastric mucosal injury and even ulcer; and IL-4 can alleviate gastritis to a certain extent, and reduce H. pylori. Colonization.
In recent years, some new Th cell subsets, such as Th17, Th22, Th9, have been found to play an important role in many chronic inflammatory diseases. Interleukin-22 is an important effector factor produced by Th17 and Th22 cells. Current reports show that IL-22 mediates inflammation by inducing keratinocytes to produce pro-inflammatory and chemokines. IL-22 is involved in the pathogenesis of skin inflammatory diseases. In Klebsiella pneumoniae and Citrobacter ronatus infection models, IL-22 is linked. Overregulation of local inflammation and stimulation of antimicrobial peptides secreted by epithelial cells are involved in host defense responses, suggesting that IL-22 plays a dual role in promoting inflammation, pathogenesis and defense protection depending on its inflammatory microenvironment. So what function does IL-22 play in H.pylori-induced infectious diseases characterized by chronic inflammation? It has not been reported yet.
[research purposes]
1. In this study, IL-22, an important effector factor of Th cells, was used to study the expression characteristics of H. pylori infection.
2. preliminarily ascertain the role and mechanism of IL-22 in H. pylori infection.
[research methods]
1. Expression characteristics of IL-22 in gastric mucosa of patients with gastritis in digestive medicine were studied. RNA was extracted from gastric mucosa, cytokine mRNA level and H.pylori colonization copy number were detected by quantitative PCR, tissue protein was extracted, IL-22 protein was detected by ELISA, gastric inflammation was detected by HE staining in pathological section. Neoxin, amoxicillin, clavulanate potassium and dysentery triple therapy were used to treat gastric mucosa. After treatment, gastric mucosa samples were collected, IL-22 expression was detected by quantitative PCR, gastric inflammation was detected by HE staining in pathological sections, and the correlation between IL-22 expression and H.pylori colonization and gastric inflammation was analyzed.
Study on the response of 2. IL-22~+ T cells to H. pylori infection
Firstly, the H.pylori 26695 infected cell model was established, and the type of IL-22 secreting cells was detected by quantitative PCR in the model of H.pylori infection, and further verified by double immunofluorescence staining and flow cytometry. Then, the response of IL-22 secreting T cell subsets in the gastric tissues of H.pylori infected patients was detected by flow cytometry. Quantitative PCR was used to identify H.pylori virulence factors inducing IL-22 expression in CagA, UreB knockout strain, H.pylori 26695 infected cell model and gastric mucosa samples.
Study on the mechanism of action of 3. IL-22 in H. pylori infection
The expression of IL-22R1 on the surface of AGS cells was detected by flow cytometry, the expression of IL-22R1 in gastric tissues was detected by quantitative PCR and tissue immunofluorescence staining, and the expression of IL-22R1 in gastric mucosa after H.pylori infection was determined. To detect the expression of IL-22 in gastric epithelial cells, detect the expression of pro-inflammatory factors and MMPs in gastric tissues by quantitative PCR, and analyze the correlation with the expression of IL-22. To explore the function of IL-22 in H.pylori infection, the chemotaxis of IL-22 on lymphocytes was detected by Transwell cell chemotaxis assay in vitro, and to explore the role of IL-22 in promoting the expression of IL-22 in gastric epithelial cells. The mechanism of inflammation.
[results]
Study on the expression characteristics of 1. IL-22 in H. pylori infection
1.1 The levels of IL-22 mRNA and protein expression in gastric mucosa of H.pylori positive patients were significantly increased (P 0.05). The results also showed that the levels of cytokines IL-17A, IL-17F and IFN-gamma mRNA related to H.pylori infection were significantly increased (P 0.05).
1.2 Further analysis showed that the level of IL-22 mRNA in gastric mucosa was positively correlated with the copy number of H.pylori (r = 0.404, P 0.01), and the severity of gastritis was classified according to HE staining of pathological sections. The results showed that the level of IL-22 mRNA in gastric mucosa of patients with moderate and severe gastritis was significantly higher than that of patients with mild gastritis (P 0.05). The level of IL-22 mRNA in gastric mucosa of patients with severe gastritis was also significantly higher than that of normal gastric mucosa (P 0.05).
1.3 After clearance treatment, the level of IL-22 mRNA in gastric mucosa decreased significantly (P 0.05), and the degree of gastric inflammation decreased (P 0.05) after clearance of H.pylori.
Study on the response of 2. IL-22~+ T cells to H. pylori infection
2.1 H.pylori 26695 and AGS cells co-cultured for 24 hours, the cell morphology showed a significant "hummingbird-like" change; cells secreted a large number of pro-inflammatory cytokines IL-8, indicating the successful establishment of H.pylori infection cell model. H.pylori 26695 infected gastric epithelial cell lines and T cell lines detected IL-22 mRNA levels significantly increased (P 0.05), while gastric tissue double-fold (P 0.05). IL-22 protein and cell surface markers showed that both epithelial cells and CD4~+, CD8~+ cells could secrete IL-22. The results suggested that gastric epithelial cells and T cells were the cell types secreting IL-22 in gastric tissues after H. pylori infection.
2.2 Flow cytometry was used to detect the subtypes of IL-22 secreted in gastric mucosa: CD3~+ cell circle gates, CD4~+ and CD8~+ cell subgroups could secrete IL-22, mainly secreted by memory T cells. The ratio of IL-22~+CD4~+, IL-22~+CD8~+, IL-17~+CD4~+, IL-17~+CD8~+, IFN-gamma~+CD4~+ cells in gastric mucosa of H.pylori infected patients was significantly higher than that of uninfected patients (P 0.05), but there was no significant difference in the ratio of IFN-gamma~+CD8~+ cells between the two groups (P 0.05). The co-expression ratio of IL-22~+IL-17~+ and IL-22~+IFN-gamma~+ in gastric mucosa of patients with H.pylori infection was significantly higher than that of patients without H.pylori infection (P 0.05).
2.3 The gastric epithelial cell lines and T cell lines were infected with CagA or UreB knockout strains and H.pylori 26695 respectively. The results showed that the level of IL-22 mRNA in CagA knockout strains was significantly lower than that in H.pylori 26695 infected strains (P 0.05), but there was no significant change in UreB knockout strains. The results of CagA gene detection showed that the level of IL-22 mRNA in gastric tissues of CagA gene positive patients with H.pylori infection was significantly higher than that of CagA gene negative patients with H.pylori infection (P 0.05), but there was no difference in the level of IL-22 mRNA between CagA gene negative patients with H.pylori infection and those without H.pylori infection. IL-22 molecules expressed in tissues.
Study on the mechanism of action of 3. IL-22 in H. pylori infection
3.1 The expression of IL-22R1 on the surface of AGS cells stimulated by cytokines and recombinant proteins was detected by flow cytometry. The results showed that IL-22R1 expression did not change significantly after AGS cells were stimulated by cytokines and recombinant proteins. The expression of IL-22R1 was positive in gastric tissues. The results of quantitative PCR showed that the expression of IL-22R1 mRNA in gastric mucosa of H.pylori positive patients was significantly increased (P 0.05), suggesting that H.pylori infection could up-regulate the expression of IL-22R1 in gastric epithelial cells.
3.2 IL-22 stimulated gastric epithelial cell line 24 hours later, the levels of S100A8, S100A9, IL-8, MMP-1, MMP-10 mRNA were significantly increased (P 0.05); at the same time, the levels of IL-8, S100A8, S100A9 mRNA in gastric tissues of H.pylori infected patients were also significantly increased (P 0.05), while the levels of MMP-1, MMP-10 mRNA were not significantly changed; correlation analysis showed that the expression of IL-22 in gastric mucosa was significantly increased. The expression of IL-22 was positively correlated with the expression of S100A8, S100A9 and IL-8 (P 0.05), but not with the expression of MMP-1 and MMP-10.
3.3 The chemotactic effect of IL-22 on lymphocytes was detected by Transwell cell chemotaxis assay. The results showed that IL-22 alone could not chemotactic lymphocytes, but could chemotactic lymphocytes in the Transwell compartment after co-culture with gastric epithelial cells for 24 hours (P 0.05). The number of chemotactic lymphocytes decreased significantly after adding neutral antibody to IL-8. The results suggest that IL-22 may be involved in inflammatory reaction by inducing gastric epithelial cells to produce chemokines such as IL-8.
[Conclusion]
1. CagA~+H.pylori infection could induce the up-regulation of IL-22 transcription and protein expression, and IL-22 expression was significantly correlated with H.pylori copy number and mucosal inflammation.
2. The secreting cells of IL-22 in gastric tissue were mainly epithelial cells and memory T lymphocytes, and the secreting T lymphocyte subsets of IL-22 were enhanced in gastric mucosa of patients with H. pylori infection.
3. In the process of H. pylori infection, IL-22 participates in the inflammatory process by regulating the expression of various pro-inflammatory factors and inducing inflammatory cell infiltration.
[meaning]
A thorough study of the function and regulation of IL-22 after H.pylori infection will help us to understand the immune response and pathogenesis of H.pylori infection more comprehensively, and provide a more sufficient theoretical basis for the prevention and treatment of H.pylori-related diseases.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R378
本文编号：2178425
[Abstract]:Helicobacter pylori (H. pylori) is a spiral, microaerobic gram-negative bacilli, mainly colonized in human gastric mucosa, has caused infection in more than 50% of the world's population. H. pylori infection is an important pathogenic factor of chronic gastritis, peptic ulcer and gastric mucosa-associated lymphoid tissue lymphoma (MALT), and gastric cancer. H. pylori infection can induce strong natural and acquired immune response, but the immune response after natural infection can not eliminate bacteria, but because of the persistent infection of bacteria, gastric mucosal immune pathological damage. So far, H. pylori chronic persistent sexuality. The mechanism of infection and related immunity is not clear.
Cytokines, as messenger molecules between cells, produce specific biological effects by binding to receptors on target cells. Chronic infection of H.pylori is the result of interaction between bacteria and host. Many cytokines participate in and play different roles in this process. It has been proved that CD4~+T cells (Th cells) are sensitive to H.pylori. Therefore, the current research on cytokines stimulated by H. pylori infection is mostly focused on Th cell-related cytokines. Previous studies have shown that IFN-gamma is mainly involved in the host inflammatory response, leading to gastric mucosal injury and even ulcer; and IL-4 can alleviate gastritis to a certain extent, and reduce H. pylori. Colonization.
In recent years, some new Th cell subsets, such as Th17, Th22, Th9, have been found to play an important role in many chronic inflammatory diseases. Interleukin-22 is an important effector factor produced by Th17 and Th22 cells. Current reports show that IL-22 mediates inflammation by inducing keratinocytes to produce pro-inflammatory and chemokines. IL-22 is involved in the pathogenesis of skin inflammatory diseases. In Klebsiella pneumoniae and Citrobacter ronatus infection models, IL-22 is linked. Overregulation of local inflammation and stimulation of antimicrobial peptides secreted by epithelial cells are involved in host defense responses, suggesting that IL-22 plays a dual role in promoting inflammation, pathogenesis and defense protection depending on its inflammatory microenvironment. So what function does IL-22 play in H.pylori-induced infectious diseases characterized by chronic inflammation? It has not been reported yet.
[research purposes]
1. In this study, IL-22, an important effector factor of Th cells, was used to study the expression characteristics of H. pylori infection.
2. preliminarily ascertain the role and mechanism of IL-22 in H. pylori infection.
[research methods]
1. Expression characteristics of IL-22 in gastric mucosa of patients with gastritis in digestive medicine were studied. RNA was extracted from gastric mucosa, cytokine mRNA level and H.pylori colonization copy number were detected by quantitative PCR, tissue protein was extracted, IL-22 protein was detected by ELISA, gastric inflammation was detected by HE staining in pathological section. Neoxin, amoxicillin, clavulanate potassium and dysentery triple therapy were used to treat gastric mucosa. After treatment, gastric mucosa samples were collected, IL-22 expression was detected by quantitative PCR, gastric inflammation was detected by HE staining in pathological sections, and the correlation between IL-22 expression and H.pylori colonization and gastric inflammation was analyzed.
Study on the response of 2. IL-22~+ T cells to H. pylori infection
Firstly, the H.pylori 26695 infected cell model was established, and the type of IL-22 secreting cells was detected by quantitative PCR in the model of H.pylori infection, and further verified by double immunofluorescence staining and flow cytometry. Then, the response of IL-22 secreting T cell subsets in the gastric tissues of H.pylori infected patients was detected by flow cytometry. Quantitative PCR was used to identify H.pylori virulence factors inducing IL-22 expression in CagA, UreB knockout strain, H.pylori 26695 infected cell model and gastric mucosa samples.
Study on the mechanism of action of 3. IL-22 in H. pylori infection
The expression of IL-22R1 on the surface of AGS cells was detected by flow cytometry, the expression of IL-22R1 in gastric tissues was detected by quantitative PCR and tissue immunofluorescence staining, and the expression of IL-22R1 in gastric mucosa after H.pylori infection was determined. To detect the expression of IL-22 in gastric epithelial cells, detect the expression of pro-inflammatory factors and MMPs in gastric tissues by quantitative PCR, and analyze the correlation with the expression of IL-22. To explore the function of IL-22 in H.pylori infection, the chemotaxis of IL-22 on lymphocytes was detected by Transwell cell chemotaxis assay in vitro, and to explore the role of IL-22 in promoting the expression of IL-22 in gastric epithelial cells. The mechanism of inflammation.
[results]
Study on the expression characteristics of 1. IL-22 in H. pylori infection
1.1 The levels of IL-22 mRNA and protein expression in gastric mucosa of H.pylori positive patients were significantly increased (P 0.05). The results also showed that the levels of cytokines IL-17A, IL-17F and IFN-gamma mRNA related to H.pylori infection were significantly increased (P 0.05).
1.2 Further analysis showed that the level of IL-22 mRNA in gastric mucosa was positively correlated with the copy number of H.pylori (r = 0.404, P 0.01), and the severity of gastritis was classified according to HE staining of pathological sections. The results showed that the level of IL-22 mRNA in gastric mucosa of patients with moderate and severe gastritis was significantly higher than that of patients with mild gastritis (P 0.05). The level of IL-22 mRNA in gastric mucosa of patients with severe gastritis was also significantly higher than that of normal gastric mucosa (P 0.05).
1.3 After clearance treatment, the level of IL-22 mRNA in gastric mucosa decreased significantly (P 0.05), and the degree of gastric inflammation decreased (P 0.05) after clearance of H.pylori.
Study on the response of 2. IL-22~+ T cells to H. pylori infection
2.1 H.pylori 26695 and AGS cells co-cultured for 24 hours, the cell morphology showed a significant "hummingbird-like" change; cells secreted a large number of pro-inflammatory cytokines IL-8, indicating the successful establishment of H.pylori infection cell model. H.pylori 26695 infected gastric epithelial cell lines and T cell lines detected IL-22 mRNA levels significantly increased (P 0.05), while gastric tissue double-fold (P 0.05). IL-22 protein and cell surface markers showed that both epithelial cells and CD4~+, CD8~+ cells could secrete IL-22. The results suggested that gastric epithelial cells and T cells were the cell types secreting IL-22 in gastric tissues after H. pylori infection.
2.2 Flow cytometry was used to detect the subtypes of IL-22 secreted in gastric mucosa: CD3~+ cell circle gates, CD4~+ and CD8~+ cell subgroups could secrete IL-22, mainly secreted by memory T cells. The ratio of IL-22~+CD4~+, IL-22~+CD8~+, IL-17~+CD4~+, IL-17~+CD8~+, IFN-gamma~+CD4~+ cells in gastric mucosa of H.pylori infected patients was significantly higher than that of uninfected patients (P 0.05), but there was no significant difference in the ratio of IFN-gamma~+CD8~+ cells between the two groups (P 0.05). The co-expression ratio of IL-22~+IL-17~+ and IL-22~+IFN-gamma~+ in gastric mucosa of patients with H.pylori infection was significantly higher than that of patients without H.pylori infection (P 0.05).
2.3 The gastric epithelial cell lines and T cell lines were infected with CagA or UreB knockout strains and H.pylori 26695 respectively. The results showed that the level of IL-22 mRNA in CagA knockout strains was significantly lower than that in H.pylori 26695 infected strains (P 0.05), but there was no significant change in UreB knockout strains. The results of CagA gene detection showed that the level of IL-22 mRNA in gastric tissues of CagA gene positive patients with H.pylori infection was significantly higher than that of CagA gene negative patients with H.pylori infection (P 0.05), but there was no difference in the level of IL-22 mRNA between CagA gene negative patients with H.pylori infection and those without H.pylori infection. IL-22 molecules expressed in tissues.
Study on the mechanism of action of 3. IL-22 in H. pylori infection
3.1 The expression of IL-22R1 on the surface of AGS cells stimulated by cytokines and recombinant proteins was detected by flow cytometry. The results showed that IL-22R1 expression did not change significantly after AGS cells were stimulated by cytokines and recombinant proteins. The expression of IL-22R1 was positive in gastric tissues. The results of quantitative PCR showed that the expression of IL-22R1 mRNA in gastric mucosa of H.pylori positive patients was significantly increased (P 0.05), suggesting that H.pylori infection could up-regulate the expression of IL-22R1 in gastric epithelial cells.
3.2 IL-22 stimulated gastric epithelial cell line 24 hours later, the levels of S100A8, S100A9, IL-8, MMP-1, MMP-10 mRNA were significantly increased (P 0.05); at the same time, the levels of IL-8, S100A8, S100A9 mRNA in gastric tissues of H.pylori infected patients were also significantly increased (P 0.05), while the levels of MMP-1, MMP-10 mRNA were not significantly changed; correlation analysis showed that the expression of IL-22 in gastric mucosa was significantly increased. The expression of IL-22 was positively correlated with the expression of S100A8, S100A9 and IL-8 (P 0.05), but not with the expression of MMP-1 and MMP-10.
3.3 The chemotactic effect of IL-22 on lymphocytes was detected by Transwell cell chemotaxis assay. The results showed that IL-22 alone could not chemotactic lymphocytes, but could chemotactic lymphocytes in the Transwell compartment after co-culture with gastric epithelial cells for 24 hours (P 0.05). The number of chemotactic lymphocytes decreased significantly after adding neutral antibody to IL-8. The results suggest that IL-22 may be involved in inflammatory reaction by inducing gastric epithelial cells to produce chemokines such as IL-8.
[Conclusion]
1. CagA~+H.pylori infection could induce the up-regulation of IL-22 transcription and protein expression, and IL-22 expression was significantly correlated with H.pylori copy number and mucosal inflammation.
2. The secreting cells of IL-22 in gastric tissue were mainly epithelial cells and memory T lymphocytes, and the secreting T lymphocyte subsets of IL-22 were enhanced in gastric mucosa of patients with H. pylori infection.
3. In the process of H. pylori infection, IL-22 participates in the inflammatory process by regulating the expression of various pro-inflammatory factors and inducing inflammatory cell infiltration.
[meaning]
A thorough study of the function and regulation of IL-22 after H.pylori infection will help us to understand the immune response and pathogenesis of H.pylori infection more comprehensively, and provide a more sufficient theoretical basis for the prevention and treatment of H.pylori-related diseases.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R378
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