主动免疫干预TGF-β信号通路对慢性哮喘模型气道炎症反应及重塑的影响

发布时间：2018-04-21 00:03

本文选题：哮喘 + 气道重塑　；参考：《北京协和医学院》2016年博士论文

【摘要】：哮喘是一个重要的全球性疾病,具有不断增加的发病率与死亡率。近年来,国内哮喘发病率尤其是在儿童当中呈急剧上升趋势。因此,加强哮喘致病机理尤其是慢性特征发展相关的机理研究,并探讨新的治疗手段与靶点,对促进哮喘临床治疗能力的提高具有重要意义。TGF- β被认为与哮喘疾病的发生、发展密切相关。一方面,TGF- β与炎症应答密切相关,其同时具有抗炎症作用与促炎症作用。另一方面,TGF- β是纤维化等组织重塑过程中的核心分子。临床研究显示,TGF- β基因多态性与哮喘疾病易感性相关联；其表达水平与疾病进程密切相关；临床分离的成纤维细胞经TGF- β刺激可大量表达细胞外基质。动物模型研究中TGF- β的外源给予、转基因或病毒载体介导的过表达、基因敲出、单克隆抗体阻断等研究,同样揭示了TGF- β在哮喘疾病机理中潜在的重要角色。然而,由于各个研究所采用的检测手段、动物模型及干预手段等存在不同,所获结论极为矛盾。目前来说,TGF- β的角色尤其是慢性哮喘气道重塑中的作用以及其靶向干预临床治疗潜能尚不明确。本项目拟应用抗细胞因子疫苗主动免疫这一新的疾病干预策略,研究TGF- β在哮喘病理机制中的可能角色,探讨其对气道炎症以及哮喘慢性应答特征如纤维化、粘液过度产生、持续性气道高反应性的作用及可能涉及的机制。同时,评价疫苗策略在疾病机制研究与临床治疗中的应用前景。本研究通过生物信息学方法预测了3个可能的TGF- β抗原肽表位,经基因重组克隆于乙肝核心抗原(HBcAg)优势表位,在大肠杆菌中获得了高效表达,并以病毒样颗粒(VLPs)形式存在。在不使用传统佐剂的情况下,呈现TGF- β抗原肽的HBcAg VLPs (HBcAg/TGF- β)有效诱导了TGF- β特异抗体应答。研究探讨了不同抗原剂量以及佐剂对免疫应答的影响。体外中和实验显示抗血清中和了TGF- β活性；体内实验表明,HBcAg/TGF- β VLPs免疫有效抑制了二甲基硝铵诱导的大鼠肝纤维化。在卵清白蛋白(OVA)诱导的急性气道过敏性炎症模型中,预防性的HBcAg/TGF- β1以及HBcAg/TGF- β2 VLPs免疫抑制了TGF- β信号传导关键分子pSmad2/3的水平,下调了支气管肺泡灌洗液(BALF)中TGF-β活性；提升了OVA刺激的BALF Th2炎性细胞因子水平；促进血清OVA-IgE的产生；此外,其下调了BALF炎性细胞浸润,但未对肺组织炎症及气道杯状细胞增生产生显著影响。在慢性模型中,HBcAg/TGF- β 1 VLPs的免疫同时增强OVA特异IgG2a与IgG1抗体应答；显著促进血清OVA特异的IgE产生；促进BALF炎性细胞因子的持续性积累,同时促进脾细胞表达Thl细胞因子IFN-γ及Th2细胞因子IL-5；显著恶化了持续性的肺组织炎症、气道杯状细胞增生及气道高反应；另一方面,HBcAg/TGF-β 1 VLPs免疫显著抑制了气道上皮下胶原沉积,降低了肺组织α-平滑肌肌动蛋白以及Ⅰ型胶原蛋白(α1)的基因表达,提示有效抑制了OVA刺激引起的肺纤维化与平滑肌增生。本研究结果显示抗细胞因子疫苗主动免疫成功干预了TGF- β 1活性,弱化了气道上皮下胶原沉积,但恶化了持续的气道炎症、粘液过度产生和气道高反应性。结果强烈提示,TGF- β 1在气道过敏性炎症应答中的角色是复杂的,取决于具体的疾病条件及免疫病理微环境。当TGF- β 1或它的拮抗剂被考虑作为潜在的疾病治疗手段时,安全性问题需要认真评价。本研究为深入了解急性和慢性哮喘病理机制的复杂性,揭示TGF- β的可能作用,以及为评价靶向TGF- β免疫调控策略在哮喘临床治疗中的应用潜能提供了重要信息。
[Abstract]:Asthma is an important global disease with increasing morbidity and mortality. In recent years, the incidence of asthma in China is increasing, especially in children. Therefore, the mechanism of strengthening the pathogenesis of asthma, especially the development of chronic characteristics, is studied, and new therapeutic methods and targets are explored to promote the clinical practice of asthma. .TGF- beta is considered to be closely related to the occurrence and development of asthma. On the one hand, TGF- beta is closely related to the inflammatory response, and it has the role of anti-inflammatory and proinflammatory. On the other hand, TGF- beta is the core molecule in the remodeling of tissues such as fibrosis. Clinical studies show that the TGF- beta gene is a beta gene. Polymorphism is associated with susceptibility to asthma; the level of expression is closely related to the process of disease; the clinical isolation of fibroblasts can express a large number of extracellular matrix through TGF- beta stimulation. In animal model studies, the exogenous administration of TGF- beta, transgene or viral vector mediated overexpression, gene knocking, monoclonal antibody blocking, and so on, It also reveals the potential role of TGF- beta in the pathogenesis of asthma. However, because of the differences in the detection methods, animal models and interventions used in various studies, the conclusions are very contradictory. At present, the role of TGF- beta is especially the role of the airway remodeling in chronic asthma and the potential therapeutic potential of its target intervention. It is not clear. This project intends to apply the new disease intervention strategy of anti cell vaccine active immunization to study the possible role of TGF- beta in the pathological mechanism of asthma and to explore the role and possible mechanisms of its role in airway inflammation and chronic response to asthma such as fibrosis, excessive mucus production, persistent airway hyperresponsiveness. In this study, 3 possible TGF- beta epitopes were predicted by bioinformatics. The gene was cloned by gene recombination in the dominant epitopes of hepatitis B core antigen (HBcAg), which was highly expressed in Escherichia coli and existed in the form of virus like particles (VLPs). TGF- beta antigen HBcAg VLPs (HBcAg/TGF- beta) effectively induced TGF- beta specific antibody response without the use of traditional adjuvant. The study explored the different antigen doses and the effects of the adjuvant on the immune response. In vitro neutralization experiments showed that antiserum neutralized TGF- beta activity; in vivo experiments showed that HBcAg/TGF- beta VLPs was immune. In the acute airway allergic inflammation induced by ovalbumin (OVA), the prophylactic HBcAg/TGF- beta 1 and HBcAg/TGF- beta 2 VLPs immunized the level of the TGF- beta signal transduction key molecule pSmad2/3, and the activity of TGF- beta activity in the bronchoalveolar lavage fluid (BALF) was lowered in the OVA induced acute airway allergic inflammation model. Increase the level of BALF Th2 inflammatory cytokines stimulated by OVA; promote the production of serum OVA-IgE; in addition, it downregulates the infiltration of BALF inflammatory cells, but does not significantly affect lung tissue inflammation and airway goblet cell proliferation. In the chronic model, HBcAg/TGF- beta 1 VLPs enhances OVA specific IgG2a and IgG1 antibody response; It can promote the production of OVA specific IgE in serum, promote the continuous accumulation of BALF inflammatory cytokines and promote the expression of Thl cytokine IFN- gamma and Th2 cytokine IL-5, which significantly exacerbate persistent pulmonary inflammation, airway goblet cell proliferation and airway hyperresponsiveness; on the other hand, HBcAg/TGF- beta 1 VLPs immunization is significantly inhibited. The hypodermic collagen deposition on the airway was made to reduce the gene expression of alpha smooth muscle actin and type I collagen (alpha 1) in the lung tissue, suggesting that the pulmonary fibrosis and smooth muscle proliferation induced by OVA stimulation were effectively inhibited. The results of this study showed that the active immunization of the anti cytokine vaccine interfered with the activity of TGF- beta 1 and weakened the airway epithelium. Lower collagen deposition, but exacerbates persistent airway inflammation, excessive mucus production and airway hyperresponsiveness. Results strongly suggest that the role of TGF- beta 1 in airway allergic responses is complex, depending on specific conditions and immuno pathological microenvironment. When TGF- beta 1 or its antagonist is considered as a potential treatment for Disease In order to understand the complexity of the pathological mechanism of acute and chronic asthma, this study reveals the possible role of TGF- beta and provides important information for evaluating the potential application potential of TGF- beta immunoregulation strategy in the clinical treatment of asthma.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R725.6

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