支气管上皮的基因和环境相互作用对哮喘表型的影响:哮喘发病机制的探讨

Asthma is a heterogeneous disease involving several phenotypes, which are affected by different genetic pathways. In 15 years, we have been focused on studying the basic and clinical aspects of asthma and reported several important novel findings in Chinese patients: (1) asthma or asthma phenotypes associated single nucleotide polymorphisms (SNPs) of susceptible loci identified by positional cloning approach including HLA-DR-DQ polymorphism regulating specific respone,beta 2 adrenoceptor polymorphisms association with nocturnal asthma, genetic variant in TNF alpha promoter for severe asthma, DPP10 and PHF11 SNPs in allergy status and lung function, and genes encoding vitamin D metabolism pathway for asthma overall risk; (2) predominance of lower circulating vitamin D levels in asthmatics, its significant association with lung function impairment in patients, and the beneficial effect of dietary antioxidants on lung function. Genome-wide association studies have located gene SNPs associated with asthma or asthmatic phenotypes. However, these gene SNPs have not been replicated in Chinese individuals with asthma, particularly, there lack the functional investigation clarifying the importance of these SNPs in asthmatic pathobiology. Asthma is a complex disorder made up of interacting local airway genetic and environmental factors through epigenetic mechanism, thus, asthma is more recognized as an airway epithelial defective disease. We will take this unique opportunity to systemically explore SNPs of genes expressing in airway epithelium and regulating asthmatic airway inflammation by adopting high-throughout DNA chips in a well-phenotyped asthma cohort (including 1000 asthma patients and 500 healthy controls) that has established and described elsewhere by us. With the pivotal aid of updated biostatistical software, we will aim at identifying new gene SNPs or haplotypes in airway mediating asthma susceptibility or asthma phenotypes, as listed in this proposal. Furthermore, we will define the functional characterizations of these gene SNPs under the specific environmental context in influencing the chronicity and persistence of asthma. The ultimate goal of this proposal is hopefully expecting to find the novel therapeutic targets that could influence the natural course of asthma (for example, supplementing with vitamin D).

哮喘是异质性疾病包括很多表型，受到不同基因调节。我们一直进行哮喘基础和临床研究，报道了（1）我国哮喘及表型相关的易感基因SNP；（2）哮喘患者维生素D明显低下与肺通气功能损害显著相关，饮食中抗氧化物对患者肺功能的有益作用。全基因组关联研究定位了哮喘或表型相关的基因座位，但这些结果在我国哮喘中没有得到复制，并且这些哮喘易感基因缺乏功能研究。支气管上皮的基因与环境因素相互作用通过表观遗传机制介导了哮喘的发病，哮喘"气道上皮缺欠性疾病"。此次申请，我们将在已经建立了表型特征完整的哮喘研究队列（1000名患者和500名对照者）中，采用定制SNP芯片的方法系统研究支气管上皮基因以及哮喘气道炎症基因SNP，通过遗传学分析，以期发现新的调节哮喘及表型的SNP或单倍型，并深入研究这些易感基因SNP与特定环境因素相互作用在哮喘发病及进展中的特点。主要目的是探索新的影响哮喘自然过程的治疗靶位如补充维生素D。

近年哮喘被认为气道上皮疾病，围绕哮喘气道上皮的特点，我们建立了哮喘研究队列包括679名哮喘个体和669名健康对照者。对表达于气道上皮的IRAK-M进行了测序，在该区域内的2个SNPs即rs16224395和rs1370128哮喘的易感性相关，并且rs2870784与外周血Eos相关，rs2141709与通气功能障碍相关。上述SNP的功能研究显示：rs1624395调节了哮喘患者外周血单核细胞IRAK-M mRNA的表达，并且IRAK-M rs1624395 等位基因G与核转录因子C-Jun结合能力明显增强。..我们采用IRAK-M基因敲除小鼠研究了IRAK-M在卵蛋白（OVA）诱发的急慢性哮喘中的作用，发现（1）急性哮喘模型IRAK-M基因敲除小鼠受到OVA致敏和刺激后气道炎症程度明显加重，支气管上皮来源的胸腺基质淋巴细胞生成素，IL-33和Th17水平显著升高，肺组织中Th17明显聚集，肺组织DC细胞表面OX40L显著上调，IRAK-M基因敲除细胞促进了CD4+T细胞向Th2或Th17转化； IRAK-M通过激活抗原递呈细胞功能，而影响了T细胞的分化而哮喘急性气道炎症。（2）OVA诱导的慢性哮喘模型与急性模型相反，IRAK-M基因敲除小鼠则呈现较轻的气道炎症和较低气道反应性，气道中Th2细胞因子水平明显降低，而Th1细胞因子水平明显升高，我们的机制研究发现IRAK-M基因敲除小鼠受到OVA慢性刺激使树突细胞表达共抑制分子PD-L1和PD-L2水平明显升高，体外分离骨髓来源的树突细胞，并以LPS刺激后，IRAK-M基因缺失的树突细胞表达Th1细胞因子的水平明显升高。我们完成了另一个研究表明，在不同强度烟雾刺激下，IRAK-M通过调节肺树突细胞和气道巨噬细胞表达不同的共刺激刺激分子，而影响了T细胞的分化，而起到了对气道的保护（急性期）或损伤（慢性期）作用。.空气污染对哮喘患者全身炎症和循环中微粒子具有影响，我们的数据表明，在2008年奥运会（严格环境控制）后2个月循环中TNF,，IL-6，IL-8，IL-10和内皮衍生的微粒子和脂多糖结合蛋白水平仍然显著下降；体外实验显示，哮喘患者的血循环中的微颗粒囊对激肽的舒张反应明显下降，对乙酰胆碱的收缩作用呈异常反应。这些数据表明药物结合环境控制能明显降低哮喘患者的炎症反应和以及内皮系统损伤。