PTEN-PI3K/Akt/mTOR通路调控自噬对Wnt经典通路诱导MSC成肺泡上皮分化的作用机制研究

Diffuse alveolar epithelial cells injury is the major pathology of ARDS. Effective repair of damage alveolar epithelial has a key role to the treatment and the improvement the prognosis of ARDS. Our previous experiments have confirmed the activation of canonical Wnt pathway promoted exogenous mesenchymal stem cells (MSC) to homing into lung tissue and differentiation into lung epithelial cells in acute respiratory distress syndrome (ARDS) mice. Many studies found that the autophagy involved in differentiation of MSC, and the canonical Wnt pathway can regulate the PTEN-PI3K/Akt/mTOR patheway which is the key regulators of the autophagy. We hypothesize that canonical Wnt pathway promotes the differentiation of MSC into lung epithelial cells through regulation of autophagy and the PTEN-PI3K/Akt/mTOR pathway. In this study, MSC is co-cultured with lipopolysaccharide induced lung epithelial cells, then the regulation of the autophagy and PTEN-PI3K/Akt/mTOR pathway are observed during the differentiation of MSC into lung epithelial cells induced by activation of canonical Wnt pathway . To assay the effect of PTEN-PI3K/Akt/mTOR pathway regulated through gene modification mediated by lentivirus transfection on the homing and differentiation of MSC, and the lung injury repair after LPS challenged in mice.It may illustrate that the effect of activation of canonical Wnt pathway on promotion of differentiation of MSC into lung epithelial cells is achieved by regulating the autophagy through regulation of the PTEN-PI3K/Akt/mTOR pathway. This study may provide an evidence for lung epithelium repair in ARDS and facilitate the further improvement of MSC-based treatment for ARDS.

弥漫性肺泡上皮损伤是ARDS的基本病理改变，有效修复损伤肺泡上皮是治疗ARDS的关键。我们已证实Wnt经典通路能促进外源性MSC向ARDS小鼠损伤肺组织归巢并分化为肺泡上皮细胞。因自噬在MSC定向分化中发挥关键作用，而Wnt经典通路能调节自噬相关的重要通路PTEN-PI3K/Akt/mTOR，推测Wnt经典通路通过PTEN-PI3K/Akt/mTOR通路调控自噬促进MSC向肺泡上皮定向分化。本研究基于MSC和内毒素损伤的肺细胞共培养模型，通过观察调控自噬及PTEN-PI3K/Akt/mTOR通路对MSC向肺泡上皮分化的影响，明确Wnt经典通路调控自噬促进MSC向肺泡上皮定向分化的分子机制；通过复制小鼠ARDS模型，给予PTEN/mTOR基因转染的MSC，观察其对MSC在肺内归巢、分化及肺上皮修复的影响，证实Wnt经典通路促进MSC分化通过PTEN-PI3K/Akt/mTOR通路调控自噬实现

弥漫性肺泡上皮细胞损伤是急性呼吸窘迫综合征（ARDS）的基本病理改变，有效修复损伤肺泡上皮是治疗ARDS的关键。我们既往的研究已证实Wnt经典通路能促进外源性间充质干细胞（MSC）向ARDS小鼠损伤肺组织归巢并分化为肺泡上皮细胞。而调节MSC分化过程中的自噬平衡是决定MSC的分化效能的关键。明确经典Wnt通路促进MSC向肺泡上皮细胞分化的细胞自噬及分子机制，对提高分化效能并从根本上提高MSC对ARDS的治疗效果具有重要意义。.本研究在体外肺上皮（MLE-12）细胞共培养结合小气道培养基（SAGM）孵育诱导小鼠MSC向Ⅱ型肺泡上皮细胞（ATⅡ）细胞分化的模型基础上，通过氯化锂（LiCl）或DKK 1分别激活或抑制经典Wnt通路，观察经典Wnt通路诱导MSC向ATⅡ细胞分化过程中细胞自噬及PTEN-PI3K/Akt/mTOR 通路相关蛋白的表达影响。结果发现经典Wnt通路促进MSC在体外向ATⅡ细胞的分化过程中，MSC细胞自噬增加以及自噬相关通路PTEN-PI3K/Akt/mTOR被激活。.本研究进一步通过慢病毒介导的干扰mTOR基因转染，构建长期稳定调控PI3K/Akt/mTOR通路的MSC细胞株，在LPS诱导的ARDS小鼠模型基础上，通过观察干扰mTOR基因的MSC在ARDS小鼠肺内细胞自噬水平，向Ⅱ型肺泡上皮细胞分化以及肺损伤修复，明确细胞自噬及PI3K/Akt/mTOR通路对MSC在ARDS小鼠肺内向Ⅱ型肺泡上皮细胞分化及肺损伤修复的调节作用。结果发现干扰mTOR基因转染活化PI3K/Akt/mTOR通路能增加MSC在ARDS小鼠体内的细胞自噬水平，促进MSC向ATⅡ细胞分化；增加小鼠肺组织上皮紧密连接蛋白的表达改善肺上皮通透性，从而促进mMSCs修复ARDS肺损伤。.本研究通过体内外实验证实，ARDS时经典Wnt通路能增加MSC的细胞自噬，活化自噬信号通路PTEN-PI3K/Akt/mTOR，并促进MSC分化为ATⅡ细胞，改善肺上皮通透性，从而促进MSC修复ARDS肺损伤，为阐明经典Wnt通路对MSC在ARDS肺泡上皮修复和结构重建中的具体分子及信号机制提供新的依据，为ARDS的细胞治疗提供新的思路。