糖皮质激素反应性复合纳米载体驱动miRNA-138表达以抑制哮喘小鼠气道重塑的研究

Epithelial mesenchymal transition (EMT) plays the important role in airway remodeling of asthma. MicroRNA-138(miR-138) can inhibit the expression of ROCK2, RhoC, ZEB2, EZH2 and Vimentin, restrain the EMT process. Increasing the expression of miR-138 is expected to reverse EMT and inhibit airway remodeling. Inhaled corticosteroids can act in airway epithelial cells. Inserting the glucocorticoid reaction element(GRE) into the upstream of the promoter of miR-138 expression vector become "glucocorticoid reaction enhancer".Inhaled corticosteroid can activate the glucocorticoid reaction enhancer, further increase the expression of the miR-138, inhibit or reverse EMT in the airway epithelial cells, and inhibit airway remodeling of asthma. Chitosan could have the effect of slow-release, protect the nucleic acid from destruction, and targeted act. The present study is to construct the composite slow-release chotisan nanoparticle that coats the plasmid containing GRE and miR-138 gene (pGRE - miR-138) and inhaled corticosteroids, absorbs the airway epithelial specific antibody on the surface of chitosan nanoparticles. This composite slow-release chotisan nanoparticle targets on airway epithelial cells. We will investigate whether the composite slow-release chotisan nanoparticle can reverse EMT phenotype of airway epithelial cells, promote the treatment sensitivity of inhaled corticosteroids and suppress airway remodeling of asthma.

吸入糖皮质激素（GC）是临床哮喘治疗的基本策略，但上皮－间质转分化(EMT)导致哮喘气道重塑机制，继而引起GC治疗不敏感以至哮喘进行性恶化。miR-138能阻滞多种ETM相关分子表达，抑制EMT，从而抑制气道重构。利用GC能通过受体进入细胞，作用于并活化下游分子的"GC反应元件（GRE）"进而驱动相关基因表达的原理，本研究拟先构建GRE驱动miR-138表达的质粒载体（pGRE-miR138），进而制备内部包封pGRE-miR138质粒和GC，表面包被气道上皮特异性抗体的复合壳聚糖纳米缓释微粒，通过体外转染或在体雾化吸入的方式，让此复合纳米载体靶向进入小鼠气道上皮。这不仅实现GC能靶向富集于气道上皮细胞，而且在细胞内，GC能活化共转染入的pGRE-miR138质粒的GRE，驱动miR-138转录，逆转EMT，恢复气道上皮对GC敏感性，抑制气道重塑，增强GC控制哮喘的疗效，并探讨其机制。

上皮间质转分化(EMT)导致哮喘气道重塑机制，吸入激素对哮喘气道重塑疗效不佳。miR-138能阻滞多种EMT相关分子表达，抑制EMT，从而抑制气道重构。利用吸入激素能通过受体进入细胞，作用于并活化下游分子的"糖皮质激素反应元件（GRE）"进而驱动相关基因表达的原理，本研究构建GRE驱动miR-138表达的质粒载体（pGRE-miR138），进而制备内部包封pGRE-miR138质粒和吸入激素的复合壳聚糖纳米缓释微粒，GC能活化共转染入的pGRE-miR138质粒的GRE，驱动miR-138转录，逆转EMT，恢复气道上皮对吸入激素敏感性，抑制气道重塑，增强吸入激素治疗哮喘的疗效。本课题成功构建“糖皮质激素反应元件GRE”和驱动的miR-138表达的质粒载体（pGRE- miR-138），制备了包封pGRE-miR-138质粒和吸入糖皮质激素的复合壳聚糖纳米缓释微粒。体外试验部分发现：TGF-β1成功诱导气道上皮细胞EMT模型，pGRE-miR-138+激素组复合壳聚糖纳米缓释微粒可部分逆转气道上皮细胞EMT，使间质细胞表型蛋白Vimentin和α-SMA蛋白表达明显下调，I型胶原蛋白表达下调，上皮细胞表型蛋白E-cadherin蛋白表达上调，并下调Smad3表达。体内试验部分：成功构建小鼠哮喘气道重塑模型，pGRE-miR-138+激素组复合壳聚糖纳米缓释微粒滴鼻至哮喘气道重塑小鼠后，哮喘小鼠的气道炎症和气道重塑均明显减轻，支气管肺泡灌洗液中IL-5、IL-13水平明显降低，INF-γ水平无明显变化，肺组织上皮细胞表型蛋白E-cadherin蛋白表达上调，肺组织间质细胞表型蛋白Vimentin、α-SMA蛋白和I型胶原蛋白表达均下调，并下调Smad3蛋白表达。以上体内外试验表明pGRE-miR-138质粒和吸入糖皮质激素的复合壳聚糖纳米缓释微粒可能通过抑制TGF-β1/Smad3通路逆转EMT，从而减轻气道重塑。