脂氧素对急性肺损伤肺泡上皮细胞-间质转化（EMT）的影响及机制研究

ALI/ARDS is a major cause of respiratory failure in critically ill patients leading to considerable morbidity and mortality. The Pathological changes seen in ALI/ARDS comprise alveolar capillary damage, disruption of the alveolar epithelium. The alveolar epithelium is populated by two epithelial cell types in normal adult lungs, alveolar epithelial type I (ATI) and ATII cells. ATII cells have many known functions, including the production, secretion and reuptake of pulmonary surfactant and surfactant proteins, and regulation of alveolar fluid in normal lungs and during the resolution of pulmonary oedema. The degree of alveolar epithelial injury is an important predictor of the outcome such as duration of mechanical ventilation and survival. Alveolar repair, such as occurs during recovery after ALI, is believed to involve the transdifferentiation of ATII cells which retain stem cell like properties into ATI cells via intermediate cell phenotypes. EMT is a process by which epithelial cells lose fundamental epithelial characteristics such as tight junctions, apical: basolateral polarity and the expression of epithelial-specific markers and assume a mesenchymal phenotype, expressing a variety of mesenchymal markers and acquiring functional characteristics of fibroblasts and myofibroblasts such as ECM production, motility and the ability to invade surrounding tissues. Therefore the regulation of EMT versus effective alveolar cell transdifferentiation may be an important determinant of the switch between an appropriate lung repair programme vs. aberrant lung remodelling and resultant airway/alveolar fibrosis. Lipoxins were the first mediators recognized to have dual anti-inflammatory and pro-resolution activities, which have been described as the endogenous “braking signal” for inflammation. Our previous research shows that the lipoxin concentration and lipoxin receptor expression decrease in ARDS patients. Lipoxin can prevent EMT in alveolar epithelium and promote ATⅡ differentiation. Base on the finding, we speculate that disorder of lipoxin is the important mechanism of acute lung injury. The main aim of this grant is to find the mechanism of lipoxin on EMT in alveolar epithelium. Lipoxin maybe used as potential novel therapeutic agent for ARDS through preventing EMT in alveolar epithelium ..

肺泡上皮主动修复是急性肺损伤失控性炎症消退的重要机制。脂氧素是机体最重要内源性抗炎促消退介质,为炎症反应刹车信号。在前期证实 ARDS 病人脂氧素浓度和受体表达明显下调基础上，结合我们新近发现脂氧素抑制肺泡上皮细胞发生EMT,并促进其转化为肺泡Ⅰ型上皮细胞等线索,我们推测:脂氧素生成或信号转导障碍可能是ALI发病的重要机制。为此，我们拟采用培养原代ATⅡ、建立大鼠ALI模型以及收集ALI病人肺泡灌洗液、血液，应用Realtime-PCR、流式细胞仪、高效液相-质谱联用、western blot 等技术,观察EMT的程度与临床预后是否具有相关性；研究脂氧素对肺泡Ⅱ型上皮细胞发生EMT的影响并探讨具体机制。进而在大鼠肺损伤模型，应用脂氧素受体激动剂和siRNA,进一步验证结果。为探寻ALI防治新靶标提供依据。

肺泡上皮主动修复是急性肺损伤失控性炎症消退的重要机制。脂氧素是机体最重要内源性抗炎促消退介质,为炎症反应刹车信号。为此，我们拟采用培养原代ATⅡ、收集ALI病人肺泡灌洗液，应用Realtime-PCR、形态学检测、western blot 等技术，我们检测脂氧素对 TGF-β 诱导人原代肺泡Ⅱ 型上皮细胞 EMT 发生形态学的影响。检测脂氧素对 TGF-β 诱导人及大鼠原代肺泡Ⅱ型上皮细胞 SP-C、 AQP5、 E-cadherin、 snail、Fibronectin、 N-cadherin、 α -SMA 等标记表达水平的影响。接着我们应用相关的信号通路阻滞剂或激动剂研究脂氧素对 TGF-β 诱导人原代肺泡Ⅱ型上皮细胞 EMT 发生的胞内信号分子 （ Smad2/3、 ERK、 PI3-K）及其活化状态的影响。同时我们建立脂多糖诱导的急性肺损伤模型，检测脂氧素对内毒素诱导的急性肺损伤整体模型 EMT 发生的影响。 检测脂氧素对急性肺损伤模型肺组织E-cadherin、 N-cadherin、 α -SMA 等标记表达水平的影响。 我们从体外及体内实验同时证实了脂氧素能抑制肺泡Ⅱ型上皮细胞 EMT 发生,并探讨其具体机制。我们证实脂氧素通过Smad及PI3-K信号通路，并以脂氧素受体依赖形式抑制TGF-β诱导肺泡上皮细胞发生EMT，并促进其转化为肺泡Ⅰ型上皮细胞。为探寻ALI防治新靶标提供依据。

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