Hippo信号通路调控间充质干细胞向ARDS肺泡上皮细胞分化的机制研究

Mesenchymal stem cells（MSC）could home to the injured lung and differentiate to alveolar epithelial cells in acute respiratory distress syndrome (ARDS). However, it has been proved that the repair capacity of MSC in ARDS model is limited by the inadequacy of homing and differentiation of MSC to alveolar epithelial cells. Thus it is important to elucidate the mechanism which regulates the homing and differentiation of MSC in order to improve the lung-protective effects of MSC in ARDS. Recent researches have indicated that Hippo signaling may play important roles in the migration and differentiation of MSC, however, the precise effects of Hippo signaling in ARDS remains to be clarified. Our pilot study also indicates that Hippo signaling is involved in the repair process of alveolar epithelial cells in early ARDS. From above all we assumed that Hippo signaling could ameliorate lung injury of ARDS through enhancing the homing and differentiation of MSC to alveolar epithelial cells. In vitro studies the damaged lung cells of ARDS model will be co-cultured with MSC under different cultural environment, whereas the expression of key signaling molecules of Hippo signaling, the proliferation, invasion and differentiation of MSC will be observed. Thereafter, in vivo modulation of Hippo signaling will be achieved by transfected MSC with LATS1/LATS1 shRNA sequence, whereas the homing and differentiation of MSC, the function of alveolar epithelial cells and lung injury pathology will be observed. Given that modulation of Hippo signaling has provided a feasible approach controlling the migration and differentiation of MSC, modulation of Hippo signaling may optimize stem cell therapy for patients with ARDS.

急性呼吸窘迫综合征（ARDS）时外源性间充质干细胞（MSC）可向肺部归巢并分化为肺上皮细胞，修复肺损伤。但MSC归巢和分化能力不足限制了其对ARDS修复效应。研究表明Hippo信号通路是调控MSC迁移和分化的关键途径，可能促进MSC对ARDS的修复，但目前无相关研究。据此假设Hippo通路可促进MSC归巢和分化为肺泡上皮修复肺损伤。本研究首先体外复制ARDS损伤肺细胞模型并与MSC共培养，比较不同诱导环境和Hippo通路功能状态对其关键信号分子表达，MSC增殖、侵袭力及向肺上皮细胞分化的影响，探讨ARDS时Hippo通路对MSC归巢和分化调控机制；继而向MSC转染LATS1/LATS1 shRNA序列调控Hippo通路功能并移入ARDS小鼠体内，观察不同归巢和分化能力的MSC修复肺泡上皮和肺损伤的作用，明确调控Hippo信号促进MSC肺保护作用有效性和可行性，为ARDS细胞治疗提供新思路。

本研究拟在ARDS小鼠模型肺单细胞悬液和MSC体外共培养模型基础上，探讨ARDS时Hippo信号通路对MSC归巢并向肺泡上皮分化的调控效应，筛选出能够增强MSC归巢和分化能力的Hippo通路功能状态，之后依据体外实验结果通过向MSC转染LATS1或LATS1 shRNA序列，将ARDS时Hippo通路调整为有利于MSC归巢并分化为肺上皮细胞的状态，阐明ARDS时调控Hippo信号通路增强MSC肺保护作用的有效性和可行性，为ARDS细胞治疗提供新的思路。本研究通过2-DG及XMU-MP-1调控Hippo信号通路，筛选出促进小鼠骨髓来源MSC(mice bone marrow derived mesenchymal stem cells, mMSCs)的分化、增殖、迁移及抗氧化应激损伤能力的Hippo通路火花状态，进而通过慢病毒载体转染LATS1调控MSC Hippo信号通路活性，确认体外实验中Hippo信号通路调控的MSC分化、增殖和迁移能力的状态，最终通过复制小鼠ARDS模型，并通过气道移植慢病毒载体转染LATS1调控其Hippo信号通路mMSCs细胞系，以明确调控Hippo信号通路在体内环境中对MSC修复ARDS肺损伤能力的影响。研究表明阻断Hippo信号通路可提高mMSCs 在体外分化、增殖、迁移和耐受H2O2诱导氧化应激损伤的能力，但通过慢病毒转染低表达LATS1抑制Hippo信号通路可提高mMSCs 在体外成骨及成脂肪分化、增殖及迁移能力，而在小鼠LPS-ARDS模型中通过移植低表达LATS1基因的mMSCs可通过抑制Hippo信号通路促进MSC向损伤肺组织的归巢和向ATII细胞的分化，改善肺水肿及肺内炎症反应，减轻肺组织病理损伤和肺纤维化病变。上述研究结果提示Hippo信号通路是调控MSC在体内外分化、增殖、迁移和耐受氧化应激的能力，调控Hippo信号通路可能有助于增强mMSCs对LPS诱导小鼠ARDS的修复效应，改善临床ARDS的结局，但仍有待于进一步人体研究证实。