从miRNA调控的PMVEC通透性研究流感病毒性肺炎肺络损伤的分子机制及解毒凉血散瘀方的干预

Damage of lung collaterals prevails in the pathological change of influenza virus-induced pneumonia. Using pulmonary microvascular endothelial cell (PMVEC) as the vector of lung collaterals, the previous study demonstrated that phosphorylation of ERM played an important role in PMVEC hyperpermeability induced by influenza virus. MiRNA is a key link in gene regulation, however, the miRNAs which regulate phosphorylation of ERM and PMVEC hyperpermeability, have not been reported. Therefore, using primary cultured PMVEC as in vitro model and mice with influenza virus-induced pneumonia as in vivo model, the study screens the differential expressional miRNAs induced by influenza virus and TNF-α, respectively, by using miRNA microarray chips; predicts the target genes regulated by miRNAs by using bioinformatics software, such as Miranda, and further verifies them by using dual luciferase report vectors; takes advantage of gain-of-function research or loss-of-function research to explore the effects of the differential expressional miRNAs and their target genes on ERM phosphorylation and PMVEC hyperpermeability. Based on the regulation of miRNA on PMVEC permeability, the study firstly explores the molecular mechanism of damage of lung collaterals in influenza virus-induced pneumonia. And the research observes the effect of classical combined formula Xijiao dihuang tang and Yinqiaosan, which had detoxification, cooling blood and dissipating stasis efficacy, on miRNA-target-gene and its related signaling pathway, to explore the formula's miRNA-regulating mechanism in improving PMVEC hyperpermeability and provide experimental evidence and novel idea for its clinical application.

流感病毒性肺炎中普遍存在肺络损伤的病变，前期研究以肺微血管内皮细胞（PMVEC）作为肺络的载体，证实ERM磷酸化是流感病毒感染PMVEC通透性升高的关键环节；miRNA是基因调控的核心成分，然而，调控ERM磷酸化、PMVEC通透性升高的miRNA分子尚未报道。本课题以原代PMVEC及流感病毒性肺炎小鼠为模型，miRNA芯片筛选流感病毒、TNF-α作用后差异表达miRNA并验证；Miranda软件及双荧光素酶报告载体预测并验证其靶基因；功能获得性或缺失性研究分析miRNA及靶基因在ERM磷酸化及PMVEC通透性升高中的作用；本课题首次从miRNA调控PMVEC通透性的角度探讨流感病毒性肺炎中肺络损伤的分子机制。并从肺络论治，观察具有解毒凉血散瘀功效的经典合方犀角地黄汤合银翘散对miRNA-靶基因相关通路的影响，探讨其改善PMVEC通透性的miRNA调控机制，为其临床应用提供依据及新的思路。

流感病毒感染可在不引起内皮细胞凋亡、坏死的情况下诱导内皮细胞通透性升高；miRNA在流感病毒感染、复制中发挥重要作用。本项目探讨了参与流感病毒感染脐静脉内皮细胞通透性升高中的miRNA分子及其靶基因。通过对比两株不同H1N1流感病毒株CA07、PR8感染脐静脉内皮细胞后miRNA的差异表达，我们发现了一系列miRNA在流感病毒感染后差异表达（如has-miR-181a-5p, has-miR-138-5p, has-miR-21-5p, has-miR-29a-3p, and has-miR-200a-3p, has-miR-147b, has-miR-501-3p, has-miR-21-3p, has-miR-23a-5p, has-miR-29b-1-5p）。这些miRNAs在流感病毒感染后出现明显下调，可通过调控MLC、actin、PKC、Ca2+-CaM、Ras/Raf/MEK/ERK、ROCK等通路中的靶基因活化，参与流感病毒诱导的内皮细胞通透性升高。在两株不同流感病毒感染的不同时间点中，我们并未发现共同的特定的miRNA，因此，我们认为流感病毒感染内皮细胞后，是通过一系列miRNA，而非某一个特定miRNA的变化，参与内皮细胞通透性调控，而PKC、Ca2+-CaM、Ras/Raf/MEK/ERK、ROCK、MLC通路均参与了通透性调控。. 流感病毒感染可导致小鼠病毒性肺炎。在流感病毒感染及犀角地黄汤合银翘散（XDY）干预后，小鼠肺组织中miRNA和mRNA都有较显著变化，差异表达miRNA主要参与了TLR、Jak−STAT、MAPK、PKC、I 型干扰素、炎性小体、肿瘤坏死因子等多个信号通路的调控。犀角地黄汤合银翘散治疗流感病毒性肺炎的作用机制可能与miR-7b-5p、miR-146b-3p、miR-146b-5p、miR-147-3p、miR-155-5p、miR-223-3p、miR-34c-5p、m iR-200b-3p、miR-503-5p、miR-669a-3p、miR-351-5p及TLR、PKC、MLC、ERK/JNK-AP-1、STAT和IFN-β相关通路的调控有关。