DNMT1在哮喘气道平滑肌细胞表型转化及气道重塑中的作用与机制研究

Airway remodeling which is partly reversible in mild asthma is pathological basis of chronicity of asthma. The phenotypic switching of airway smooth muscle cells (ASMC) from a differentiated to a synthesis/proliferation one plays an important role in the occurrence and development of airway remodeling in asthma. Our previous studies indicated that DNA methyltransferase 1 (DNMT1) mediated PDGF-induced airway smooth muscle cell phenotypic switching and the switching was abrogated when DNMT1 was inhibited. So we suppose that DNMT1 plays a key role in airway remodeling in asthma.In this study,overexpression of DNMT1 mediated by recombinant adenovirus and down expression are employed to investigate the effect of DNMT1 on PDGF-induced cell contraction, proliferation, synthesis and secretion of inflammatory mediators and extracellular matrix in primary ASM cells. In vivo, a mouse model of chronic asthma is applied to explore the effect of DNMT1 on ASM cell phenotype, smooth muscle layer and deposition of extracellular matrix (ECM). On this basis, the reversal of synthesis/proliferation ASMCs and intervention of airway remodeling in asthma are to be observed by inhibiting DNMT1 to figure out the role and status of DNMT1 in asthmatic airway remodeling. DNA methylation and gene expression microarrays, immunology and molecular biology technologies are employed to explore the mechanism of DNMT1-mediated ASMC phenotypic switching to shed light on the pathogenesis of asthmatic airway remodeling from a new perspective. This study helps to provide a new way to prevent and treat asthma.

气道重塑是哮喘慢性化的病理基础，早期可部分逆转。分化型气道平滑肌细胞(ASMC)向合成/增殖型转化在哮喘气道重塑发生、发展中起重要作用。我们既往研究发现DNA甲基化转移酶1(DNMT1)介导PDGF诱导的ASMC表型转化，抑制DNMT1可阻止这一转化，据此推测DNMT1在哮喘气道重塑中扮演重要角色。本项目拟采用重组腺病毒高表达与下调表达策略，观察DNMT1对PDGF诱导的原代ASMC收缩、增殖、合成分泌炎性介质和ECM等的影响；在体观察其对慢性哮喘模型小鼠ASMC表型、气道平滑肌层和ECM沉积等的影响。在此基础上，抑制DNMT1，观察哮喘合成/增殖型ASMC表型逆转、气道重塑干预情况，以明确DNMT1在哮喘气道重塑中的作用和地位。采用甲基化芯片、基因表达谱芯片、免疫学、分子生物学等技术深入探讨DNMT1调控ASMC表型转化的作用机制,为阐明哮喘气道重塑发病机制奠定基础，为哮喘的防治提供新思路。

哮喘是一种全球范围内常见、高发，以气道炎症、气道重塑和气道高反应等为主要特征的异质性疾病。气道重塑是影响哮喘预后的重要因素，是导致难治性哮喘和哮喘严重发作的重要原因，一旦形成难以逆转，因此早期干预极为重要。本项目研究中我们证实DNA甲基化在哮喘中发挥重要作用，DNMT1在哮喘中上调表达，干预DNMT1表达水平能抑制哮喘环境下原代气道平滑肌细胞由分化成熟型向合成/增殖型转化、抑制平滑肌细胞异常增殖、迁移及合成细胞外基质能力。我们筛选出Aza-CdR腹腔注射小鼠的安全剂量，发现该剂量能通过抑制哮喘小鼠DNMT1的上调表达，减少肺组织异常甲基化；能有效干预哮喘气道炎症及杯状上皮细胞粘液的过度合成，为深入探讨哮喘气道重塑发病机制奠定基础，为哮喘的防治提供新的策略。通过分析哮喘患者与健康志愿者气道平滑肌及刷检的气道上皮细胞基因表达谱芯片，筛查出在哮喘状态下发挥重要作用的基因，为后续深入研究奠定了实验基础。表达谱芯片结果显示slit2在哮喘患者气道上皮细胞中显著降低，我们通过在体及体外对哮喘环境下slit2表达进行了验证及干预。体外，我们以屋尘螨天然有效成分Der p2刺激人气道上皮细胞株，检测slit2基因变化情况，结果表明，Der p2刺激下，Beas-2b细胞slit2时间、剂量依赖性下调，Aza-CdR预处理能抑制哮喘环境下slit2基因表达水平的降低。我们对哮喘环境下slit2下调机制进行了深入探讨，发现Slit2基因启动子CpG岛甲基化水平增加，Aza-CdR预处理能显著抑制该基因的下调表达。为进一步研究受甲基化水平调控slit2基因在哮喘气道重塑中的作用，我们采用与气道重塑关系密切且在哮喘环境高表达的TGF-β1刺激Beas-2b细胞，以外源性slit2-N进行干预，结果发现，外源性slit2-N抑制TGF-β1诱导的Beas-2b细胞向间质细胞转化、并通过抑制细胞骨架的重组降低细胞迁移能力，提示slit2有望成为干预哮喘气道重塑早期事件的潜在靶点。本项目研究结果表明，DNA甲基化转移酶抑制剂Aza-CdR对哮喘气道上皮损伤异常修复、粘液异常合成及气道平滑肌细胞异常生物学行为等具有很好的干预作用，将为哮喘气道结构重塑的早期防治提供新的思路。