BDNF基因多态性在过敏性鼻炎中的研究及上颌窦后鼻孔息肉组织病理学和炎性发病机制的研究

发布时间：2018-04-26 06:27

本文选题：脑源性神经营养因子 + 中重度过敏性鼻炎　；参考：《山东大学》2017年博士论文

【摘要】：研究背景过敏性鼻炎(Allergic rhinitis,AR)是在鼻部黏膜接触过敏原后,由特异性IgE介导的黏膜炎症反应。AR在全世界范围内的发病率约为10%～40%,不仅严重影响患者的生活质量,也给社会带来沉重的医疗负担。近年来,AR的发病率呈逐年上升的趋势。目前,AR的病因尚不完全清楚,但个体基因和环境之间复杂的相互作用是其发病的危险因素之一。在过去的几十年中,AR基因相关研究是其病因学研究的热点。脑源性神经营养因子(BDNF)是一种分泌蛋白,是神经营养因子家族中的一员。嗜酸性粒细胞是参与AR免疫反应发生及发展的重要炎症细胞,可生成BDNF蛋白并表达其相应的受体TrkB。BDNF单核苷酸多态性(SNPs)已被证实与过敏性哮喘的发病密切相关。因此,BDNF可能与AR的发病密切相关。但目前,BDNF SNPs是否会增加中国区域人群过敏性鼻炎的患病风险,以及BDNF遗传变异对BDNF基因及蛋白表达水平的影响尚不清楚。研究方法本研究选取山东地区有典型过敏症状并且过敏原检测阳性的481例中重度变应性鼻炎患者作为实验组,健康对照组1735例;选取新加坡中国人群中典型过敏症状并且过敏原检测阳性的522例中重度变应性鼻炎患者作为实验组,健康对照组717例;提取受试者外周血DNA。自HapMap中国汉族人群(CHB)中筛选出BDNF标签SNP位点,从每一样本基因组DNA中取5ng进行多重PCR扩增,通过MassARRAY系统检测标签SNP位点,对受试者进行基因分型,评估与AR患病风险的关联。最后,通过体外及体内实验对BNDF遗传变异所产生的功能效应进行验证。研究结果在山东人群及新加坡中国人群这两个独立的群体中,BDNFSNP rs10767664均与中重度变应性鼻炎有显著相关性(P=0.039,OR=1.17;P=0.0017,OR =1.324)。山东人群中风险等位基因A在AR中的出现率为57%,高于对照组的53%。通过PLINKmeta分析将以上两个独立群体整合,BDNF SNP rs10767664仍与中重度变应性鼻炎有显著相关性(P = 0.000298,OR= 1.242)。BDNF非同义SNP rs6265与SNP rs10767664连锁不平衡,遂使用rs6265进行体外功能研究。Val/Met重组质粒转染HEK293T细胞,与空载体组相比,细胞裂解液中BDNF蛋白的表达水平均显著增高。在体内,rs6265 CC基因型者外周血BDNF蛋白水平显著高于CT型及TT型(P= 0.02,P= 0.03);rs6265 CC基因型者,外周血IgE水平显著高于TT型(P0.05)。因此,rs10767664AA基因型者更易表现为中重度过敏性鼻炎,且其血浆中BDNF蛋白及总IgE水平更高。大样本量数据eQTL meta分析显示,BDNF SNPs与外周血BDNF mRNA的表达水平具有显著相关性。结论BDNF SNPs增加了中重度过敏性鼻炎的患病风险;BDNF rs10767664 AA基因型者外周血BDNF mRNA及蛋白的表达水平更高;BDNF SNP rs10767664与过敏性鼻炎的发生密切相关。研究背景上颌窦后鼻孔息肉(AntrochoanalPolyps,ACP)约占全部鼻腔息肉的4%～6%。与临床上常见的双侧鼻息肉(bilateralnasalpolyps,BNP)相比,ACP的发病年龄较低,病变范围较局限,临床症状相对于BNP也较轻。在过去的几十年中,对于传统双侧鼻息肉发生的分子机制的基础研究众多,但对ACP病理改变及发病机制的研究极少。本研究旨在较大样本量的中国人群ACP中进行较为全面的组织病理学及炎性发病机制的相关研究,明确ACP的上皮重塑模式,对组织中常见炎症细胞的浸润进行检测,分析上皮重塑及炎细胞浸润与ACP临床特点的关系,检测ACP中辅助性T细胞、调节性T细胞及其转录因子和相关炎性介质的表达,并比较其表达与对照组鼻腔黏膜的差异。研究方法收集上颌窦后鼻孔息肉ACP 33例,传统双侧鼻息肉BNP 49例,以及行鼻中隔偏曲手术的下鼻甲黏膜50例作为对照组。通过HE及IHC染色,对组织的上皮重塑及嗜酸性粒细胞、中性粒细胞、巨噬细胞、肥大细胞、CD4+T细胞、CD8+T细胞和FOXP3+T-reg细胞的浸润进行评估和比较。提取21例ACP及34例对照组的mRNA,通过逆转录及实时定量PCR,检测组织中Th1、Th2、Th17和T-reg细胞的转录因子及相关炎性介质基因水平的表达。研究结果与BNP相比,ACP的上皮增生及杯状细胞增生程度较轻。在本研究中,87.9%的后鼻孔息肉表现为中性粒细胞型鼻息肉,其中性粒细胞的浸润程度显著高于BNP及对照组:与对照组相比,ACP中巨噬细胞及CD8+ T细胞的浸润显著上调,但肥大细胞的浸润是下调的。在ACP组织中,FOXP3+调节性T细胞的浸润数目与中性粒细胞、巨噬细胞、CD4+T细胞和CD8+T细胞的浸润均呈显著正相关性;嗜酸性粒细胞的浸润程度与ACP患者的哮喘史具有显著的正相关性;巨噬细胞的浸润程度与上皮的增生程度及杯状细胞的增生程度呈显著负相关性;CD8+ T细胞的浸润程度与鳞状上皮化生呈显著正相关性。与对照组相比,调节性T细胞T-reg的相关转录因子FOXP3及炎性介质IL10在ACP中的表达显著上调。此外,IL6、中性粒细胞相关基因MPO、嗜酸性粒细胞聚集基因CCL13和CCL18在ACP中的表达显著上调,但Th1/Th2/Th17的转录因子T-bet,GATA3和RORc的表达显著下调。FOXP3的mRNA表达水平与T-bet和GATA3呈显著正相关性,但与RORc无相关性;IL6的mRNA表达水平与T-bet、GATA3和FOXP3呈显著正相关性;IL10的mRNA表达水平与T-bet呈显著正相关性;此外,MPO的mRNA表达水平与CCL18呈显著正相关性。IL10的mRNA表达水平与巨噬细胞的浸润数量呈显著正相关;MPO的mRNA表达水平与CD4+ T细胞的浸润个数呈显著负相关性,与CD8+ T细胞的浸润数量显著负相关性;T-bet的mRNA表达水平与CD8+ T细胞的浸润个数呈显著正相关性;RORc的mRNA表达水平与CD4+ T细胞的浸润数量呈显著负相关性。结论ACP的上皮重塑特点、炎细胞浸润和炎症因子表达模式与BNP有所不同;ACP中涉及复杂的炎症细胞浸润,包括显著上调的中性粒细胞、CD8+T细胞和巨噬细胞;IL6、IL10和FOXP3的mRNA的表达水平显著上调,并与其它细胞因子和炎症细胞类型的表达具有一定的相关性,可能在ACP的发病过程中起到一定的调控作用,这为进一步深入研究ACP的发病机制奠定了基础,也为ACP的诊断和治疗提供新思路。
[Abstract]:Background allergic rhinitis (Allergic rhinitis, AR) is in the nasal mucosa after exposure to allergens, and the incidence of.AR in the whole world is about 10% ~ 40%, which is mediated by specific IgE, which not only seriously affects the quality of life of the patients, but also brings heavy medical burden to the society. In recent years, the incidence of AR has been increasing year by year. At present, the etiology of AR is not yet fully understood, but the complex interaction between the individual gene and the environment is one of the risk factors for its pathogenesis. In the past few decades, AR gene related research is a hot spot in its etiology. Brain derived neurotrophic factor (BDNF) is a secretory protein and is one of the family of neurotrophic factors. Eosinophils are important inflammatory cells involved in the occurrence and development of AR immunoreaction. BDNF protein can be generated and its corresponding receptor TrkB.BDNF single nucleotide polymorphism (SNPs) has been closely related to the pathogenesis of allergic asthma. Therefore, BDNF may be closely related to the pathogenesis of AR. But, at present, whether BDNF SNPs will increase or not The risk of allergic rhinitis in the regional population and the effect of BDNF genetic variation on the expression of BDNF gene and protein is not clear. The research method selected 481 cases of moderate and severe allergic rhinitis with typical allergic symptoms and positive allergen detection in Shandong as experimental group and 1735 healthy control group. 522 moderate and severe allergic rhinitis patients with positive allergic symptoms and positive allergen detection were used as experimental group and 717 healthy control group. The BDNF tag SNP site was selected from the peripheral blood DNA. from the Chinese Han population (CHB) of HapMap, and 5ng for multiple PCR amplification was obtained from each sample genome DNA. The MassARRAY system detected the label SNP loci, genotyping the subjects and assessing the association with the risk of AR disease. Finally, the functional effects of BNDF genetic variation were verified by in vitro and in vivo experiments. The results were both in the two independent groups of the Shandong population and the Chinese population in Singapore, and BDNFSNP rs10767664 was both in and in the middle of the population. There was a significant correlation between severe allergic rhinitis (P=0.039, OR=1.17; P=0.0017, OR =1.324). The incidence of A in AR was 57% in Shandong population, which was higher than that of the control group by PLINKmeta analysis to integrate the above two independent groups. BDNF SNP rs10767664 still had a significant correlation with moderate and severe allergic rhinitis (0.000298, 1.242).BDNF unsynonymous SNP rs6265 and SNP rs10767664 were unbalance, then rs6265 was used to study in vitro function of.Val/Met recombinant plasmid transfected to HEK293T cells, and the expression level of BDNF protein in the cell lysate was significantly higher than that in the unloaded body group. In the body, the level of the peripheral blood BDNF protein in the rs6265 CC base was significantly higher than that of the type of HEK293T. TT type (P= 0.02, P= 0.03); rs6265 CC genotype, the level of IgE in peripheral blood was significantly higher than that of the TT type (P0.05). Therefore, the rs10767664AA genotype was more likely to be a medium and severe allergic rhinitis, and the BDNF protein and IgE level in the plasma were higher. Significant correlation. Conclusion BDNF SNPs increased the risk of moderate to severe allergic rhinitis; the expression level of BDNF mRNA and protein in peripheral blood of BDNF rs10767664 AA genotype was higher; BDNF SNP rs10767664 was closely related to the occurrence of allergic rhinitis. Compared with common bilateral nasal polyps (bilateralnasalpolyps, BNP), the 4% ~ 6%. of meat is lower, the scope of the disease is relatively limited and the clinical symptoms are relatively light relative to BNP. In the past few decades, there are many basic studies on the molecular mechanism of the traditional bilateral nasal polyps, but the pathological changes and pathogenesis of ACP have been studied. The study is very rare. This study aims at a relatively comprehensive study of histopathology and inflammatory pathogenesis in the large sample size of Chinese population ACP, to identify the epithelial remodeling patterns of ACP, to detect the infiltration of common inflammatory cells in the tissues, to analyze the relationship between epithelial remodeling and inflammatory cell infiltration and the clinical characteristics of ACP, and to detect the complement of ACP. T cells, regulatory T cells and their transcriptional factors and related inflammatory mediators were expressed, and the difference between the expression and the control group was compared with the nasal mucosa in the control group. The methods collected 33 cases of ACP, 49 cases of traditional bilateral nasal polyps, 49 cases of traditional bilateral nasal polyps, and 50 cases of inferior turbinate mucosa with nasal septum deflection. Through HE and IHC The epithelial remodeling of tissue and the infiltration of eosinophils, neutrophils, macrophages, mast cells, CD4+T cells, CD8+T cells and FOXP3+T-reg cells were evaluated and compared. The mRNA of 21 cases of ACP and 34 control groups was extracted, and the transcription factors of Th1, Th2, Th17, and T-reg cells in the tissues were detected by reverse transcription and real-time quantitative PCR. In this study, 87.9% of the posterior nostril polyps showed a neutrophil type nasal polyp in this study. In this study, 87.9% of the nasal polyps were neutrophilic polyps, and the degree of infiltration of granulocytes was significantly higher than that of the BNP and the control group. Compared with the control group, the macrophage and CD in ACP were compared with the control group. The infiltration of 8+ T cells was significantly up-regulated, but the infiltration of mast cells was down. In ACP tissue, the number of FOXP3+ regulatory T cells was significantly correlated with the infiltration of neutrophils, macrophages, CD4+T cells and CD8+T cells, and the Jin Runcheng degree of eosinophils had a significant positive correlation with the history of asthma in ACP patients. There was a significant negative correlation between the degree of macrophage infiltration and the degree of epithelial proliferation and the degree of goblet cell proliferation; the degree of infiltration of CD8+ T cells was positively correlated with the squamous metaplasia. Compared with the control group, the expression of the related transcription factor FOXP3 of the regulatory T cell T-reg and the expression of inflammatory mediating IL10 in ACP was significantly up-regulated. In addition, IL6 The expression of neutrophil related gene MPO, eosinophil aggregation gene CCL13 and CCL18 in ACP was significantly up-regulated, but the expression level of Th1/Th2/Th17 transcriptional factor T-bet, GATA3 and RORc was significantly lower than that of T-bet and GATA3, but there was no correlation with T-bet and GATA3. There was a significant positive correlation with FOXP3, and the level of mRNA expression in IL10 was positively correlated with T-bet, and the mRNA expression level of MPO was positively correlated with CCL18, and the mRNA expression level of.IL10 was positively correlated with the number of macrophages, and mRNA expression level of MPO was negatively correlated with the number of infiltration of CD4+ cells. There was a significant negative correlation between the number of cell infiltration and the significant positive correlation between the mRNA expression level of T-bet and the number of infiltration of CD8+ T cells; the mRNA expression level of RORc was negatively correlated with the number of CD4+ T cells. Conclusion the epithelial remodeling characteristics of ACP, inflammatory cell infiltration and the expression pattern of inflammatory factors are different from BNP; ACP involved in the recurrence of BNP. The infiltration of mixed inflammatory cells, including the significantly up-regulated neutrophils, CD8+T cells and macrophages, the expression level of mRNA in IL6, IL10 and FOXP3 is significantly up-regulated, and has a certain correlation with the expression of other cytokines and inflammatory cell types, and may play a regulatory role in the pathogenesis of the pathogenesis of ACP, which is further in-depth study. The study laid a foundation for the pathogenesis of ACP, and provided new ideas for the diagnosis and treatment of ACP.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R765.21

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