自噬相关因子Atg7对肺II型细胞功能的影响机制

Alveolar epithelial Type II cells (AT II) maintain alveolar tension by secreting surfactant proteins. AT II also has the capability of self-renewal and differentiation into alveolar epithelial Type I cells. The deficiency of surfactant proteins caused by the dysfunction of AT II can lead to the occurrence of neonatal respiratory distress syndrome (NRDS). Our recent work showed that autophagy-related gene Atg7 knockout (Atg7-/-) neonate died 12 hours after birth, due to cyanosis and respiratory distress. Further studies have shown that Atg7-/- mice have alveolar type II dysplasia, lower number of lamellar bodies and reduced surfactant proteins. In view of previous studies reported that autophagy is involved in the regulation of cellular secretion and stemness maintenance, we hypothesize that Atg7 is involved in regulating of the maturation, secretion and stemness of AT II. In this study, we employ MLE12 cells and AT II-specific Atg7 conditional knockout mice as the experimental model to study the effects of Atg7 on the formation, maturation and secretion of surfactant proteins and lamellar bodies. Acute lung injury model induced by LPS will be employed to investigate the effect of Atg7 on the damage repair of AT II.

Ⅱ型肺泡上皮细胞通过分泌表面活性物质维持肺泡张力，并通过其干细胞特性实现自我更新和向I型细胞的分化。II型细胞发育障碍所致的表面活性物质缺乏是新生儿呼吸窘迫综合征发生的重要原因。我们近期工作显示自噬相关基因Atg7敲除小鼠（Atg7-/-）12小时内因呼吸窘迫呈现缺氧性紫绀并死亡，并发现该小鼠的Ⅱ型细胞发育不良、板层小体数目减少伴表面活性物质不足。基于文献报道和前期基础，我们推测Atg7介导的自噬参与调控Ⅱ型细胞的成熟、分泌功能，通过影响II型细胞的干细胞功能调控其自我更新及肺组织器官重塑能力。本项目将以MLE12细胞及Ⅱ型肺泡细胞特异性敲除Atg7鼠为模型，结合透射电镜、ELISA及免疫印迹等方法明确Atg7对表面活性物质及板层小体形成、成熟和分泌的影响；通过LPS诱导的急性肺损伤模型探索Atg7对II型细胞损伤修复能力的影响；以期阐明Atg7通过调控II型细胞影响肺组织功能的机制。

板层小体（LB）是一种同心结构，含有表面活性蛋白和磷脂，是II型肺泡上皮细胞（AT2）的特异性细胞器。然而，LBs的起源尚未完全阐明。我们之前报道过自噬调节Weibel–Palade小体（WPB）的形成，在此我们证明自噬参与另一种溶酶体相关细胞器LB的形成。我们发现在发育过程中，LBs由含有糖原等细胞质成分的自噬体转化而来。在野生型新生小鼠中观察到LBs和自噬体之间的融合。此外，自噬活性标记物微管相关蛋白1轻链3B（LC3B）主要共同定位于LB的限制膜上。小鼠AT2细胞中的自噬相关基因7（Atg7）全敲除和AT2细胞条件性敲除均导致LB成熟和表面活性蛋白B产生缺陷。此外，自噬活性的改变调节LB的形成和表面活性蛋白B的产生。综上所述，这些结果表明，自噬在发育过程中调节LB的形成及成年期维持LB的稳态中起着关键作用。