【摘要】： 幽門螺桿菌(Helicobacter pylori,H. pylori)是一種螺旋狀、微需氧革蘭陰性桿菌,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、消化性潰瘍及胃粘膜相關(guān)淋巴組織淋巴瘤(MALT)等疾病的重要致病因子,與胃癌的發(fā)生密切相關(guān),已被WHO列為Ⅰ類致癌因子。H. pylori感染可以引起機(jī)體較強(qiáng)烈的天然和獲得性免疫應(yīng)答,但自然感染H. pylori后的免疫反應(yīng)并不能清除細(xì)菌,反而由于持續(xù)感染導(dǎo)致胃粘膜免疫病理損害。目前,H. pylori慢性持續(xù)性感染的機(jī)理仍不十分清楚。 CD4+T細(xì)胞即Th細(xì)胞在H. pylori感染的免疫應(yīng)答中發(fā)揮重要作用,Th細(xì)胞被激活后,通過不同的分化途徑分化為效應(yīng)性Th細(xì)胞,獲得特定的生物學(xué)功能,根據(jù)其產(chǎn)生細(xì)胞因子和生物學(xué)功能的不同,傳統(tǒng)上將效應(yīng)性CD4+T細(xì)胞分為Th1和Th2亞群。H. pylori感染通常表現(xiàn)為粘膜組織內(nèi)特異性CD4+T細(xì)胞增加,T細(xì)胞主要產(chǎn)生IL-2、IFN-γ、IL-12等細(xì)胞因子,提示為Thl型免疫應(yīng)答。現(xiàn)多認(rèn)為Thl型免疫應(yīng)答參與了H. pylori感染慢性炎癥的進(jìn)程,并導(dǎo)致胃粘膜損傷甚至潰瘍,但僅僅Th1型免疫應(yīng)答并不能完全解釋H. pylori感染后胃炎的發(fā)生。 近年研究發(fā)現(xiàn)一類不同于Th1和Th2細(xì)胞的Th細(xì)胞亞群,此細(xì)胞亞群分泌IL-17、IL-6,IL-22和IL-21,而不分泌IL-4和IFN-γ,被命名為Th17細(xì)胞亞群。Thl7細(xì)胞主要介導(dǎo)慢性炎癥,在自身免疫病、炎癥性疾病及防御粘膜胞外菌感染中發(fā)揮重要作用。Thl7細(xì)胞的發(fā)現(xiàn)及其分化和功能的研究,有助于我們對以慢性炎癥為機(jī)制的疾病的理解和闡明,為慢性炎癥疾病的免疫防治提供了新的思路。那么Th17細(xì)胞在H. pylori感染過程中呈現(xiàn)什么樣的應(yīng)答規(guī)律,在H. pylori感染中發(fā)揮什么樣的功能呢?目前還不清楚。 【研究目的】 1.本研究擬以Th17細(xì)胞及其主要效應(yīng)因子IL-17作為研究的主要對象,研究其在H. pylori感染中的應(yīng)答規(guī)律; 2.初步明確Th17細(xì)胞在H. pylori慢性感染中的作用及初步機(jī)制探討。 【研究方法】 1.建立H. pylori感染BALB/c小鼠模型,在H. pylori感染后不同時間點處死小鼠,取胃組織提取RNA檢測細(xì)胞因子mRNA水平變化,提取胃組織蛋白,ELISA檢測細(xì)胞因子蛋白水平變化,取小鼠的脾,腸系膜淋巴結(jié),胃旁淋巴結(jié)分離淋巴細(xì)胞后,細(xì)胞內(nèi)因子染色,FCM檢測Th1,Th2和Th17細(xì)胞應(yīng)答。 2.基因工程方法制備GST-IL-17融合蛋白,免疫家兔制備抗IL-17抗體,鑒定其中和IL-17的活性�？诜辔窰. pylori前腹腔注射抗IL-17抗體,real-time PCR檢測阻斷IL-17效應(yīng)后H. pylori定植量的變化,HE染色檢測胃組織炎癥;并采用AdmIL-17口服灌胃后感染H. pylori,檢測調(diào)控IL-17高表達(dá)對H. pylori定植量及胃組織炎癥的影響。 3.培養(yǎng)小鼠胃上皮細(xì)胞(MFC),加入IL-17刺激24 h,收集細(xì)胞,抽提RNA,real-time PCR檢測基質(zhì)金屬蛋白酶(MMP )和趨化因子的變化,并在上述IL-17阻斷和高表達(dá)模型中檢測MMP和趨化因子的變化。 【研究結(jié)果】 1. Th17細(xì)胞及相關(guān)細(xì)胞因子在H. pylori感染中的應(yīng)答規(guī)律 1.1 H. pylori感染組的小鼠胃組織中IFN-γ和IL-17的表達(dá)在mRNA及蛋白水平均顯著增高,而IL-4的表達(dá)在蛋白水平?jīng)]有顯著的變化。調(diào)控Th1和Th17細(xì)胞應(yīng)答的細(xì)胞因子IL-12和IL-23的表達(dá)在H. pylori感染后也有顯著升高。 1.2通過流式細(xì)胞技術(shù)檢測Th細(xì)胞的頻率發(fā)現(xiàn):H. pylori感染小鼠的脾淋巴細(xì)胞中H. pylori特異性的Th1和Th17細(xì)胞的比率顯著高于正常小鼠的脾淋巴細(xì)胞,而Th2細(xì)胞應(yīng)答變化不顯著。提示H. pylori感染可以誘導(dǎo)特異性的Th1和Th17細(xì)胞應(yīng)答。 1.3 Th細(xì)胞應(yīng)答呈現(xiàn)動態(tài)變化:H. pylori感染后7 d,在腸系膜淋巴結(jié)檢測到Th1,Th2及Th17細(xì)胞的應(yīng)答,在脾淋巴細(xì)胞中,Th17細(xì)胞在14 d開始增多,21 d達(dá)到高峰,Th1細(xì)胞在21 d開始增多,而Th2細(xì)胞在整個感染進(jìn)程中沒有顯著變化。在胃旁淋巴結(jié)中Th細(xì)胞的應(yīng)答較晚在28 d出現(xiàn)應(yīng)答,其Th1及Th17細(xì)胞應(yīng)答的規(guī)律與脾的情況類似。脾和胃旁淋巴結(jié)的淋巴細(xì)胞中Th細(xì)胞應(yīng)答顯示Th17細(xì)胞應(yīng)答要早于Th1細(xì)胞應(yīng)答,并且不同淋巴器官中Th細(xì)胞應(yīng)答的時間提示腸系膜淋巴結(jié)可能是H. pylori激發(fā)特異性免疫應(yīng)答較早的場所。 2. Th17/IL-17促進(jìn)H. pylori的定植以及胃組織炎癥 2.1兔抗IL-17抗體處理組的小鼠H. pylori定植量及胃組織炎癥顯著均顯著低于對照IgG處理組小鼠(P 0.05)。結(jié)果提示中和IL-17的活性有助于H. pylori的清除并減輕炎癥,說明Th17/IL-17途徑可能削弱了抗H. pylori感染的宿主防御。 2.2我們采用AdmIL-17感染胃組織上調(diào)IL-17的表達(dá)后再感染H. pylori,檢測H. pylori定植量和胃組織炎癥的變化。AdmIL-17處理小鼠,與AdLuc和PBS處理組相比,在H. pylori感染后顯著增加H. pylori的定植量(P 0.01),而AdLuc處理組與PBS處理組相比,H. pylori的定植量沒有顯著變化(P 0.05)。HE染色切片顯示,AdmIL-17組小鼠胃組織炎癥程度強(qiáng)于AdLuc處理組(P 0.0125),而AdLuc處理組小鼠的胃粘膜炎癥與PBS組相比沒有顯著性變化(P 0.05)。這些結(jié)果顯示Th17 / IL-17可能增加了宿主對H. pylori感染的敏感性。 3. Th17細(xì)胞/IL-17在H. pylori感染中的作用機(jī)制初探 3.1小鼠胃上皮細(xì)胞系MFC細(xì)胞在IL-17的刺激下,MMP-2,MMP-3,MMP-7,MMP-9和趨化因子CCL2,CCL5,CCL20,CCL25以及CXCL1等mRNA表達(dá)與未刺激的細(xì)胞相比均顯著增高(P 0.05)。提示MMP和趨化因子可能參與了H. pylori感染中IL-17介導(dǎo)的免疫效應(yīng)。 3.2 IL-17抗體處理后H. pylori感染胃組織中MMP-9和CCL25的表達(dá)與對照IgG處理小鼠相比顯著降低(P 0.05),而其他MMP和趨化因子的表達(dá)沒有顯著變化。 3.3 AdmIL-17處理后H. pylori感染組,與AdLuc和PBS處理組相比,MMP-2, MMP-9和CCL25的表達(dá)顯著增高(P 0.05)。以上結(jié)果提示我們Th17細(xì)胞及IL-17可能通過誘導(dǎo)MMP-9以及CCL25的表達(dá)參與調(diào)控H. pylori感染及炎癥反應(yīng)。 【主要結(jié)論】 1. H. pylori感染后可以誘導(dǎo)H. pylori特異性的Th1和Th17細(xì)胞應(yīng)答。Th17細(xì)胞應(yīng)答要早于Th1細(xì)胞應(yīng)答,并且不同淋巴器官中Th細(xì)胞應(yīng)答的時間提示腸系膜淋巴結(jié)可能是H. pylori激發(fā)特異性免疫應(yīng)答較早的場所。 2. Th17/IL-17可以促進(jìn)H. pylori感染后的細(xì)菌定植以及炎癥的程度。 3. Th17/IL-17可能是通過調(diào)控MMP以及趨化因子的表達(dá)參與H. pylori感染后的細(xì)菌定植以及炎癥的調(diào)控。 【研究意義】 Th17/IL-17可能成為H. pylori感染相關(guān)疾病輔助治療的靶標(biāo),深入了解H. pylori感染后Th細(xì)胞免疫應(yīng)答特征,功能及其調(diào)控,將有助于我們更全面了解H. pylori感染的致病機(jī)制,并且有助于指導(dǎo)H. pylori相關(guān)免疫疾病的免疫治療。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R378

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 張藍(lán)方;CD4+CD25+調(diào)節(jié)T細(xì)胞和Th17細(xì)胞在特發(fā)性血小板減少性紫癜中的表達(dá)和意義[D];濟(jì)南大學(xué);2011年

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本文編號：2238664