Th17细胞及IL-17在幽门螺杆菌感染中的功能研究
发布时间：2018-09-12 10:00
【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,HE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)。HE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。
[Abstract]:Helicobacter pylori (H. pylori) is a spiral, microaerobic gram-negative bacilli, mainly colonized in human gastric mucosa, has caused more than 50% of the world's population infection. H. pylori infection is a chronic gastritis, peptic ulcer and gastric mucosa-associated lymphoid tissue lymphoma (MALT) and other important pathogenic factors, and the occurrence of gastric cancer. The infection of H. pylori can induce strong natural and acquired immune responses, but the immune response after natural infection of H. pylori can not eliminate bacteria. On the contrary, the gastric mucosal immune pathological damage is caused by persistent infection. At present, the mechanism of chronic persistent infection of H. pylori is still not clear. Very clear.
CD4+T cells, or Th cells, play an important role in the immune response to H.pylori infection. Th cells are activated and differentiated into effector Th cells through different differentiation pathways to obtain specific biological functions. According to their cytokine production and biological functions, effector CD4+T cells are traditionally divided into Th1 and Th2 subgroups. It is generally believed that Thl-type immune response participates in the process of chronic inflammation of H.pylori infection and leads to gastric mucosal injury and ulcer, but only Th1-type immune response can not be completed. Totally explain the occurrence of gastritis after H. pylori infection.
Recent studies have found a class of Th cell subsets different from Th1 and Th2 cells, which secrete IL-17, IL-6, IL-22 and IL-21, but do not secrete IL-4 and IFN-gamma. Thl7 cells are named Th17 cell subsets. Thl7 cells mainly mediate chronic inflammation and play an important role in autoimmune diseases, inflammatory diseases and defense against mucosal extracellular bacterial infections. The discovery, differentiation and function of Th17 cells in H. pylori infection will help us understand and clarify the mechanism of chronic inflammation and provide new ideas for the prevention and treatment of chronic inflammatory diseases. It's not clear.
[research purposes]
1. Th17 cells and its main effector IL-17 were selected as the main objects of this study to study their response to H. pylori infection.
2. preliminarily clarify the role and preliminary mechanism of Th17 cells in chronic infection of H. pylori.
[research methods]
1. To establish the BALB/c mice model infected by H. pylori, the mice were sacrificed at different time points after H. pylori infection. The levels of cytokine mRNA were detected by RNA extraction from gastric tissues, the levels of cytokine protein were detected by ELISA, and the cytokines were extracted from spleen, mesenteric lymph nodes and paragastric lymph nodes. Th1, Th2 and Th17 cell responses were detected by staining and FCM.
2. GST-IL-17 fusion protein was prepared by genetic engineering and immunized rabbits to prepare anti-IL-17 antibody and identify the activity of anti-IL-17. Anti-IL-17 antibody was injected intraperitoneally before oral administration of H.pylori. Pylori, the effects of IL-17 overexpression on H. pylori colonization and gastric tissue inflammation were detected.
3. Cultured mouse gastric epithelial cells (MFC) were stimulated with IL-17 for 24 hours. Cells were collected, RNA was extracted, and the changes of matrix metalloproteinase (MMP) and chemokines were detected by real-time PCR. The changes of MMP and chemokines were detected in the above IL-17 blocking and overexpressing models.
[results]
1. the response of Th17 cells and related cytokines in H. pylori infection
1.1 The expression of IFN-gamma and IL-17 in gastric tissues of mice infected with H.pylori increased significantly at mRNA and protein levels, while the expression of IL-4 did not change significantly at protein level. The expression of cytokines IL-12 and IL-23 regulating the responses of Th 1 and Th17 cells also increased significantly after H.pylori infection.
1.2 The frequency of Th cells detected by flow cytometry showed that the ratio of H.pylori-specific Th 1 and Th17 cells in splenic lymphocytes of H.pylori-infected mice was significantly higher than that of normal mice, but the response of Th2 cells was not significantly changed.
1.3 Th cell response showed a dynamic change: 7 days after H.pylori infection, Th1, Th2 and Th17 cells were detected in mesenteric lymph nodes. Th17 cells began to increase in splenic lymphocytes at 14 days, reached a peak at 21 days, Th1 cells began to increase at 21 days, while Th2 cells did not change significantly during the whole infection process. Th1 and Th17 cell responses were similar to those of the spleen. Th17 cell responses in the lymphocytes of the spleen and paragastric lymph nodes showed that Th17 cell responses were earlier than that of Th1 cells. Th cell responses in different lymphoid organs suggested that the mesenteric lymph nodes might be H. pylori-specific. A place where sexual immune response was earlier.
2. Th17/IL-17 promotes H. pylori colonization and gastric tissue inflammation.
2.1 The amount of H.pylori colonization and gastric inflammation in the rabbit anti-IL-17 antibody group were significantly lower than those in the control IgG group (P 0.05). The results suggest that the neutralization of IL-17 may contribute to the clearance of H.pylori and reduce inflammation, suggesting that Th17/IL-17 pathway may weaken the host defense against H.pylori infection.
2.2 AdmIL-17 was used to increase the expression of IL-17 in gastric tissues and then infect H.pylori. H.pylori colonization and gastric inflammation were detected. AdmIL-17 treated mice significantly increased H.pylori colonization (P 0.01) after H.pylori infection compared with AdLuc and PBS treated mice. HE staining showed that gastric inflammation in AdmIL-17 group was stronger than that in AdLuc group (P 0.0125), while gastric mucosal inflammation in AdLuc group was not significantly different from that in PBS group (P 0.05). These results suggest that Th17/IL-17 may increase the sensitivity of host to H.pylori infection.
Preliminary study on the mechanism of 3. Th17 cell /IL-17 in H. pylori infection
3.1 The expression of MMP-2, MMP-3, MMP-7, MMP-9 and chemokines CCL2, CCL5, CCL20, CCL25 and CXCL1 in gastric epithelial cell line MFC stimulated by IL-17 was significantly higher than that in non-stimulated cells (P 0.05). It suggested that MMP and chemokines might be involved in the IL-17-mediated immune response in H.pylori infection.
3.2 The expressions of MMP-9 and CCL25 in H.pylori infected gastric tissues were significantly lower than those in IgG treated mice (P 0.05), while the expressions of other MMP and chemokines were not significantly changed.
3.3 Compared with AdLuc and P BS, the expression of MMP-2, MMP-9 and CCL25 in H.pylori infected group increased significantly (P 0.05). These results suggest that Th17 cells and IL-17 may participate in the regulation of H.pylori infection and inflammation by inducing the expression of MMP-9 and CCL25.
[main conclusions]
1.H.pylori infection can induce H.pylori-specific Th1 and Th17 cell responses.
2. Th17/IL-17 can promote bacterial colonization and inflammation after H. pylori infection.
Th17/IL-17 may be involved in the regulation of bacterial colonization and inflammation after H.pylori infection by regulating the expression of MMP and chemokines.
[research significance]
Th17/IL-17 may be the target of adjuvant therapy for H.pylori-related diseases. A thorough understanding of the characteristics, functions and regulation of Th cell immune response after H.pylori infection will help us to understand the pathogenesis of H.pylori infection more comprehensively and guide the immunotherapy 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】：2009
【分类号】：R378
本文编号：2238664
[Abstract]:Helicobacter pylori (H. pylori) is a spiral, microaerobic gram-negative bacilli, mainly colonized in human gastric mucosa, has caused more than 50% of the world's population infection. H. pylori infection is a chronic gastritis, peptic ulcer and gastric mucosa-associated lymphoid tissue lymphoma (MALT) and other important pathogenic factors, and the occurrence of gastric cancer. The infection of H. pylori can induce strong natural and acquired immune responses, but the immune response after natural infection of H. pylori can not eliminate bacteria. On the contrary, the gastric mucosal immune pathological damage is caused by persistent infection. At present, the mechanism of chronic persistent infection of H. pylori is still not clear. Very clear.
CD4+T cells, or Th cells, play an important role in the immune response to H.pylori infection. Th cells are activated and differentiated into effector Th cells through different differentiation pathways to obtain specific biological functions. According to their cytokine production and biological functions, effector CD4+T cells are traditionally divided into Th1 and Th2 subgroups. It is generally believed that Thl-type immune response participates in the process of chronic inflammation of H.pylori infection and leads to gastric mucosal injury and ulcer, but only Th1-type immune response can not be completed. Totally explain the occurrence of gastritis after H. pylori infection.
Recent studies have found a class of Th cell subsets different from Th1 and Th2 cells, which secrete IL-17, IL-6, IL-22 and IL-21, but do not secrete IL-4 and IFN-gamma. Thl7 cells are named Th17 cell subsets. Thl7 cells mainly mediate chronic inflammation and play an important role in autoimmune diseases, inflammatory diseases and defense against mucosal extracellular bacterial infections. The discovery, differentiation and function of Th17 cells in H. pylori infection will help us understand and clarify the mechanism of chronic inflammation and provide new ideas for the prevention and treatment of chronic inflammatory diseases. It's not clear.
[research purposes]
1. Th17 cells and its main effector IL-17 were selected as the main objects of this study to study their response to H. pylori infection.
2. preliminarily clarify the role and preliminary mechanism of Th17 cells in chronic infection of H. pylori.
[research methods]
1. To establish the BALB/c mice model infected by H. pylori, the mice were sacrificed at different time points after H. pylori infection. The levels of cytokine mRNA were detected by RNA extraction from gastric tissues, the levels of cytokine protein were detected by ELISA, and the cytokines were extracted from spleen, mesenteric lymph nodes and paragastric lymph nodes. Th1, Th2 and Th17 cell responses were detected by staining and FCM.
2. GST-IL-17 fusion protein was prepared by genetic engineering and immunized rabbits to prepare anti-IL-17 antibody and identify the activity of anti-IL-17. Anti-IL-17 antibody was injected intraperitoneally before oral administration of H.pylori. Pylori, the effects of IL-17 overexpression on H. pylori colonization and gastric tissue inflammation were detected.
3. Cultured mouse gastric epithelial cells (MFC) were stimulated with IL-17 for 24 hours. Cells were collected, RNA was extracted, and the changes of matrix metalloproteinase (MMP) and chemokines were detected by real-time PCR. The changes of MMP and chemokines were detected in the above IL-17 blocking and overexpressing models.
[results]
1. the response of Th17 cells and related cytokines in H. pylori infection
1.1 The expression of IFN-gamma and IL-17 in gastric tissues of mice infected with H.pylori increased significantly at mRNA and protein levels, while the expression of IL-4 did not change significantly at protein level. The expression of cytokines IL-12 and IL-23 regulating the responses of Th 1 and Th17 cells also increased significantly after H.pylori infection.
1.2 The frequency of Th cells detected by flow cytometry showed that the ratio of H.pylori-specific Th 1 and Th17 cells in splenic lymphocytes of H.pylori-infected mice was significantly higher than that of normal mice, but the response of Th2 cells was not significantly changed.
1.3 Th cell response showed a dynamic change: 7 days after H.pylori infection, Th1, Th2 and Th17 cells were detected in mesenteric lymph nodes. Th17 cells began to increase in splenic lymphocytes at 14 days, reached a peak at 21 days, Th1 cells began to increase at 21 days, while Th2 cells did not change significantly during the whole infection process. Th1 and Th17 cell responses were similar to those of the spleen. Th17 cell responses in the lymphocytes of the spleen and paragastric lymph nodes showed that Th17 cell responses were earlier than that of Th1 cells. Th cell responses in different lymphoid organs suggested that the mesenteric lymph nodes might be H. pylori-specific. A place where sexual immune response was earlier.
2. Th17/IL-17 promotes H. pylori colonization and gastric tissue inflammation.
2.1 The amount of H.pylori colonization and gastric inflammation in the rabbit anti-IL-17 antibody group were significantly lower than those in the control IgG group (P 0.05). The results suggest that the neutralization of IL-17 may contribute to the clearance of H.pylori and reduce inflammation, suggesting that Th17/IL-17 pathway may weaken the host defense against H.pylori infection.
2.2 AdmIL-17 was used to increase the expression of IL-17 in gastric tissues and then infect H.pylori. H.pylori colonization and gastric inflammation were detected. AdmIL-17 treated mice significantly increased H.pylori colonization (P 0.01) after H.pylori infection compared with AdLuc and PBS treated mice. HE staining showed that gastric inflammation in AdmIL-17 group was stronger than that in AdLuc group (P 0.0125), while gastric mucosal inflammation in AdLuc group was not significantly different from that in PBS group (P 0.05). These results suggest that Th17/IL-17 may increase the sensitivity of host to H.pylori infection.
Preliminary study on the mechanism of 3. Th17 cell /IL-17 in H. pylori infection
3.1 The expression of MMP-2, MMP-3, MMP-7, MMP-9 and chemokines CCL2, CCL5, CCL20, CCL25 and CXCL1 in gastric epithelial cell line MFC stimulated by IL-17 was significantly higher than that in non-stimulated cells (P 0.05). It suggested that MMP and chemokines might be involved in the IL-17-mediated immune response in H.pylori infection.
3.2 The expressions of MMP-9 and CCL25 in H.pylori infected gastric tissues were significantly lower than those in IgG treated mice (P 0.05), while the expressions of other MMP and chemokines were not significantly changed.
3.3 Compared with AdLuc and P BS, the expression of MMP-2, MMP-9 and CCL25 in H.pylori infected group increased significantly (P 0.05). These results suggest that Th17 cells and IL-17 may participate in the regulation of H.pylori infection and inflammation by inducing the expression of MMP-9 and CCL25.
[main conclusions]
1.H.pylori infection can induce H.pylori-specific Th1 and Th17 cell responses.
2. Th17/IL-17 can promote bacterial colonization and inflammation after H. pylori infection.
Th17/IL-17 may be involved in the regulation of bacterial colonization and inflammation after H.pylori infection by regulating the expression of MMP and chemokines.
[research significance]
Th17/IL-17 may be the target of adjuvant therapy for H.pylori-related diseases. A thorough understanding of the characteristics, functions and regulation of Th cell immune response after H.pylori infection will help us to understand the pathogenesis of H.pylori infection more comprehensively and guide the immunotherapy 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】：2009
【分类号】：R378
【引证文献】
相关硕士学位论文 前1条
1 张蓝方;CD4+CD25+调节T细胞和Th17细胞在特发性血小板减少性紫癜中的表达和意义[D];济南大学;2011年
，本文编号：2238664
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