Midkine-AngII信号通路调控呼吸机相关肺损伤及修复作用的机制研究

Mechanical ventilation is necessary to sustain life in patients with ARDS but the mechanical strain or shear stress generated during mechanical ventilation can induce severe injury and insufficient epithelial repair mechanisms resulting in the development of lung fibrosis. Epithelial to mesenchymal transition(EMT) plays an important role in the progression of pulmonary fibrosis. The novel cytokine midkine(MK) can promote the proliferation, differentiation of cells and has been implicated in the pathogenesis of tissue fibrosis in other settings. We observed that mechanical stretch could induce EMT in lung epithelial cells mediated by increased expression of Midkine. However, it remains unclear as to the exact regulatory mechanisms .AngII has been reported to mediate EMT in other cells and related to lung injury.However it remains unclear as to the exact role of AngII in mediating the MK induced EMT in lung epithelial cells .The project intends to use cell distraction instrument to stretch pulmonary epithelial cells and to establish the model of mechanical stretch induced EMT in vitro， make animal experiment in vivo and do the clinical research. And application Western blot, co-immunoprecipitation, RT-PCR and SiRNA to detect the expression levels of EMT-related key protein molecules to explore the the role of Midkine-AngII signaling on ventilator-induced lung injury and repair. It could provide a scientific basis and theoretical molecular drug targets for the better prevention of ventilator-induced lung injury.

机械通气是治疗ARDS重要治疗手段，然而不适当的通气可引起肺上皮细胞损伤，甚至修复过度发生纤维增殖。上皮间质转分化是纤维增殖形成的重要机制之一,中期因子（Midkine, MK）是新近发现可调节细胞增殖分化的重要因子。在我们前期研究中发现机械牵张可通过激活MK信号通路诱发肺上皮细胞转分化，然而关于其确切发生机制目前仍不十分清楚。已知AngII与细胞分化增殖及肺损伤密切相关，而其在肺上皮细胞受牵张后转分化的调控作用目前还未阐明。本课题在前期研究的基础上拟通过体外肺上皮细胞牵张模型及动物通气实验以及临床研究相结合，利用基因检测结合蛋白质分析检测方法，首次观察拮抗血管紧张素II后，机械通气对肺上皮细胞转分化标志物及MK表达的影响，阐释Midkine-AngII信号通路在调控呼吸机相关肺损伤及修复的作用机制，为更好防治呼吸机相关性肺损伤提供科学依据及可能的药物新靶点。

机械通气是目前重症呼吸疾病重要有效的治疗支持手段之一，但不适当地机械通气可使原有的肺损伤加重，导致呼吸机相关性肺损伤（Ventilator-induced lung injury, VILI）。因此如何最大程度减少呼吸机相关肺损伤，发挥呼吸机的真正保护作用至关重要。尽管过去进行了大量的研究，关于VILI确切发生机制目前还未完全阐明。如何更好地发挥肺保护作用对于危重病患者救治意义重大。深入研究机械通气对肺上皮细胞损伤及修复的影响，有助于明确VILI发生的分子机制。我们的前期研究表明过度机械牵张可以通过中期因子Midikine促使肺上皮发生间质转化。在本项目资助下，项目负责人按照研究计划开展研究工作并取得系列研究成果：1、成功构建了体外机械牵张促进肺上皮细胞转分化模型；2、过度机械牵张可以增加TGF-β1、波形蛋白及I型胶原以及AngII纤维化相关基因表达；3、Midkine在机械牵张诱导的上皮细胞间质转化中起关键作用；沉默Midkine的表达可以抑制机械牵张介导的的肺上皮间质转分化；4、初步探讨了Midkine-AngII参与机械牵张诱导肺上皮细胞上皮间质转分化的潜在分子机制；5、ARDS患者肺泡灌洗液中Midkine，AngII的浓度相较于健康志愿者增高，Midkine, AngII的浓度与疾病严重严重程度呈正相关。此外我们还进一步完善了本单位ARDS患者临床资料数据库和扩大了ARDS临床标本库，并进一步研究了体外膜肺在减轻重症患者呼吸机相关肺损伤中的作用以及IL-33及IL-36在呼吸机相关肺损伤的临床意义。本项目共发表SCI收录文章1篇，发表中文核心期刊文章2篇。待发表SCI文章3篇；项目研究成果参加国内外学术交流3次。通过本项目的研究我们初步探讨了Midkine-AngII 信号通路在调控呼吸机相关肺损伤和修复的关键作用及分子机制，并阐明了体外膜肺在减轻呼吸机相关肺损伤的作用，为呼吸机相关肺损伤的诊疗提供了新的方法，分子标记物和治疗靶点。