Scinderin调节气道上皮细胞骨架重排在哮喘气道重塑中的作用及机制研究

Airway remodeling is a critical reason that made asthma difficult to be cured, and there is no effective means for its early diagnosis and treatment. The dysfunction of airway epithelium cells (AECs) was implicated in the occurrence and development of airway remodeling. Our previous studies demonstrated that cytoskeleton rearrangement was associated with airway remodeling in asthma. Recently, we found that scinderin up-regulated in the AECs of asthmatic mice and was involved in epithelial-mesenchymal transition (EMT) in AECs. Based on these results, we supposed that scinderin might play a critical role in airway remodeling in asthma. This study was conducted to explore the relationship between the expression of scinderin in AECs and the airway remodeling in asthmatic mice. Regulated expressions of scinderin were applied to observe the effect on the structures and function of AECs and the hyperplasia of airway smooth muscle cells induced by AECs in order to clarify its role in the occurrence and development of airway remodeling in asthma. Cytoskeleton inhibitors and activators as well as signal pathway inhibitors were applied to investigate the mechanism by which scinderin regulated airway remodeling using immunology and molecular biology technology and this research will provide a novel strategy for treating airway remodeling with early stage. To analyze the possibility of scinderin in the shed AECs from the induced sputum as a diagnosis of the occurrence of airway remodeling in asthmatic patients ,and it might provide a new idea for the early diagnosis and treatment of asthmatic airway remodeling.

气道重塑是哮喘难以治愈的重要原因，尚无早期诊治的有效手段。气道上皮细胞（AECs）结构和功能异常是哮喘气道重塑的始动环节和持续进展的诱因。我们既往研究表明细胞骨架重排参与哮喘气道重塑，前期研究又发现肌切蛋白scinderin在哮喘AECs中上调表达且参与气道上皮-间质转化，据此推测scinderin在哮喘气道重塑中极为重要。本项目拟分析哮喘小鼠肺组织中scinderin的表达与气道重塑的关系；调控scinderin基因表达观察其对哮喘小鼠AECs结构和功能、气道平滑肌增生的影响，明确其在哮喘气道重塑发生发展中的作用；利用细胞骨架抑制剂、激活剂、信号通路抑制剂等，采用免疫学、分子生物学等技术深入探讨scinderin调控哮喘气道重塑的作用机制，为哮喘气道重塑的干预提供新的策略；分析诱导痰中脱落AECs 中scinderin蛋白表达水平作为早期诊断哮喘气道重塑的可能性，为哮喘气道重塑早期诊治提供新思路。

气道重塑是哮喘的重要病理特征，一旦形成便持续进展且难以逆转，是导致难治性哮喘和哮喘严重发作的重要诱因，可发生在哮喘早期阶段，因此，早发现、早干预对于有效防治哮喘极为重要。气道上皮损伤-修复是哮喘气道重塑的始动环节，而气道上皮细胞骨架重排及细胞极性改变在哮喘气道上皮损伤修复及气道重塑过程中扮演重要角色。我们采用鸡卵白蛋白（OVA）和屋尘螨（HDM）建立哮喘模型，证实SCIN异常高表达与哮喘密切相关，有望成为哮喘早期预测标志物。采用SCIN基因敲除小鼠建立慢性哮喘模型，结果发现SCIN基因敲除能抑制哮喘小鼠气道上皮细胞杯状化生、气道上皮细胞E-cadherin的异常分布、减轻上皮下胶原纤维及平滑肌层增厚等气道重塑特征。这些结果表明敲除SCIN基因能有效抑制哮喘小鼠气道上皮结构变化、抑制气道重塑，为临床防治哮喘气道重塑提供新的思路。体外以HDM（D.Pteronyssinus）及其主要致敏组分Der p1刺激人支气管上皮（HBE）细胞株，结果显示，HDM和 Der p1均以浓度、时间依赖性刺激HBE细胞SCIN蛋白表达。哮喘环境下，HBE去极化，而敲减SCIN基因抑制HBE的去极化改变，维持细胞的极化状态。哮喘环境下WNT5a调控HBE细胞SCIN基因表达。这些结果提示，过敏原通过WNT5a上调SCIN基因表达，后者通过调控HBE细胞骨架重排参与哮喘发生发展。分析哮喘患者外周小气道、中央气道组织标本及刷检标本基因表达谱芯片，结果发现只有KRT23和CYP2J2基因在所有纳入的哮喘样本中都与正常对照组表达差异。这些结果提示KRT23、CYP2J2与哮喘密切相关，为进一步深入研究奠定了基础。免疫组化结果证实哮喘小鼠气道上皮KRT23表达明显高于正常对照组，而体外Der p1时间依赖性上调KRT23蛋白表达。研究中我们发现哮喘小鼠肺组织中HDAC5基因的5’-UTR的CpG岛甲基化增加，而抑制DNA甲基转移酶后其异常甲基化被抑制。这一结果提示，DNA甲基化调控的组蛋白乙酰化在哮喘中发挥重要作用，为深入探讨哮喘发生机制以期早期防治哮喘提供了新的研究思路。