ORMDL3对哮喘血管重塑的影响及机制

Vascular remodeling in asthma promotes thickening of airway wall, aggravates airway hyperresponsiveness.It is of great significance for the process and prognosis of asthma..The polymorphism of ORM1-like 3 (ORMDL3) is closely related with the susceptibility and severity of asthma. ORMDL3 expression in bronchial epithelial cells increases 127 times after allergic stimulation in the wild type mice. In 2007, Moffatt found that more than one third of asthma children had differences in chromosome 17 ORMDL3 expression, which was closely related with both early-onset and late-onset asthma. It is also reported,ORMDL3 transgenic mice have obviously airway changes, including hyperplasia of airway smooth muscles, epithelial fibrosis and increased mucus secretion,also increased airway reactivity to acetyl choline armor. All these show that ORMDL3 plays an important role in chronic airway changes in asthma,while its effects on vascular remodeling are not very clear..It has been recently reported that, the expression of MMP –9 is much promoted in ORMDL3-transfected bronchial epithelial cells. MMP-9 is activated by white blood cells, degradats vascular collagen, speeds up the migration of smooth muscle cells to the matrix, induces the release of VEGF, thus promotes formation of new blood vessels.MMP-9 is closely related with VEGF in asthma.VEGF induces angiogenesis,enhances the permeability of vessel walls,and promotes MMP-9 mRNA transcription and protein secretion in smooth muscle cells. MMP-9 is also downregulated by application of VEGF receptor inhibitor in asthma mice. VEGF and MMP - 9 expression are regulated by the ERK1/2 pathway. ORMDL3 causes ERK1/2 phosphorylation and then activation of AP - 1. As one of AP-1 family members, c-Jun targets the promoter region to induce VEGF and MMP-9 expression..Our previous studies have found that there was increased angiogenesis in lung tissues in asthma mice. VEGF expression was significantly increased after 7 days OVA stimulation in early asthma, and was gradually reduced after 28 days. ORMDL3 was promoted in lung tissues and positive correlated with vessel areas in asthma mice.And it is supposed that ORMDL3 plays a role in vascular remodeling..In this research,we are to find the effects and mechanisms of ORMDL3 in asthmatic vascular remodeling using murine models of asthma and ORMDL3 transgenic mice.Then to test the influences of ORMDL3 on MMP - 9 and VEGF expression in bronchial epithelial cells by ORMDL3 shRNA and ORMDL3-overexpressing slow virus,and the influences on the proliferation, migration and tube forming of endothelial cells.Further to investigate the role of ERK1/2 in the regulation pathway.

血管重塑为哮喘慢性进展特征，显著影响预后,机制研究意义重大。血清类粘蛋白1样蛋白3（ORMDL3）是哮喘密切相关基因。ORMDL3转染人支气管上皮细胞，促进MMP-9表达。MMP-9可降解血管外基质，与VEGF分泌密切相关，二者表达受ERK1/2调控。ORMDL3对血管重塑的影响及机制尚不明确。我们前期研究发现，ORMDL3在哮喘小鼠肺组织高表达，与血管面积呈显著正相关，提示ORMDL3在哮喘血管重塑中有重要作用。本课题拟采用哮喘小鼠模型、ORMDL3转基因小鼠观察ORMDL3在哮喘血管重塑的作用；进一步通过ORMDL3过表达和shRNA慢病毒颗粒转染验证ORMDL3对支气管上皮细胞MMP-9、VEGF表达的调控及对内皮细胞增殖、迁移和管状成形能力的影响；并应用ERK1/2拮抗剂和 激动剂明确ORMDL3调控信号通路。该研究深化了哮喘血管重塑的机制理解，为临床干预提供新的靶点。

支气管哮喘是呼吸系统常见病、多发病。研究支气管哮喘进展和预后的影响因素意义重大。作为由多种细胞和细胞因子参与的气道慢性炎症性疾病，哮喘长期反复发作导致气道结构的改变，包括气道上皮下纤维化、基底膜增厚、平滑肌增生肥大、血管重塑(vascular remodeling)等。血管重塑在哮喘气道重塑中有重要作用，直接影响哮喘慢性化进程及预后。 本课题通过建立哮喘动物模型和支气管上皮细胞培养干预实验观察ORMDL3在哮喘气道重塑尤其是血管重塑中的作用。通过建立哮喘小鼠模型，应用RT-PCR、免疫组化及Western blot方法检测哮喘小鼠肺组织ORMDL3、MMP-9、p-ERK、VEGF表达，均较正常小鼠显著增高。ORMDL3、MMP-9、p-ERK、VEGF表达与肺组织血管面积及气道壁厚度正相关，且ORMDL3水平分别与MMP-9、ERK、VEGF水平正相关，提示ORMDL3通过调控ERK/MMP-9/VEGF通路促进气道重建、血管重塑。本实验首先证明了ORMDL3通过ERK/MMP-9/VEGF通路在哮喘小鼠肺组织气道重建、血管重塑中的作用，其次证明哮喘8w和12w不同时间段各因子的表达变化，进一步通过细胞刺激试验和ORMDL3shRNA和过表达慢病毒颗粒转染，验证ORMDL3对支气管上皮细胞MMP-9、VEGF表达的调控。综上所述，血管重塑是气道重建的重要组成部分，影响哮喘慢性化进程和预后的关键环节。本课题有助于帮助我们深入理解哮喘气道重建、血管重塑的病理生理机制，同时提供了新的治疗靶点。