PM2.5所致肺损伤中细胞衰老对肺泡干细胞再生修复功能的调控机制

The fine particulate Matters (PM2.5) has been demonstrated to elicit oxidative stress response, which abrogates the function of lung and other tissues, and therefore remains great threat to human health. As alveolar stem cells, alveolar type 2 (AT2) cells play a critical role in restoration of alveolar epithelium after lung injury. Excessive reactive oxygen species induce cell senescence. However, it is still unknown whether or not PM2.5 accumulation in alveolar space results in AT2 cell senescence, thereby impairing their capacity to regenerate alveolar epithelium after PM2.5 induced lung injury. In current study, by adopting whole-body PM2.5 exposure mouse model, coupled with molecular biological, cell biological and pathological technologies, we will focus on two key processes associated with PM2.5-induced lung injury, i.e. both damage and repair of alveolar epithelium through studying the correlation between PM2.5 dosage, onset of AT2 cell senescence and extent of alveolar epithelial damage. Direct effects of PM2.5 on AT2 cells will be evaluated by in vitro culture assay. We will also investigate the induction of senescence of AT2 cells by H2O2, and the effect of senescence on AT2 cell function. By doing this, we will reveal the relationship between PM2.5 concentration, oxidative stress, AT2 cell senescence and alveolar epithelial injury. This study will uncover biological mechanism underlying PM2.5 induced lung injury.

大气细颗粒物PM2.5诱导氧化应激反应，引起肺脏等多组织损伤，严重危害人群健康。2型肺泡细胞（简称AT2细胞）是肺泡干细胞，在肺泡上皮黏膜损伤后的修复中发挥重要作用。过量的活性氧自由基诱导细胞衰老。PM2.5在肺内的累积是否通过诱导AT2细胞的衰老，损害其修复肺泡上皮黏膜的能力尚未见报道。本课题以AT2细胞为研究对象，采用PM2.5全身暴露染毒小鼠模型，并分子生物学、细胞生物学和病理学技术，对PM2.5造成肺损伤的两个关键环节：肺泡上皮黏膜的损伤和修复，进行分子机制探讨。重点考查PM2.5暴露浓度与AT2细胞衰老、肺泡上皮黏膜损伤程度的相关性，研究PM2.5对AT2细胞的直接调节作用，探索活性氧自由基诱导AT2细胞衰老的发生，并细胞衰老对AT2细胞再生修复功能的影响，逐步揭示PM2.5—活性氧自由基—肺泡干细胞衰老—肺泡上皮损伤的关系机制，阐明大气雾霾细颗粒物诱发肺损伤的生物学机制。

大气细颗粒物PM2.5诱导氧化应激反应，引起肺脏等多组织损伤，严重危害人群健康。2型肺泡细胞（简称AT2细胞）是肺泡干细胞，在肺泡上皮黏膜损伤后的修复中发挥重要作用。过量的活性氧自由基诱导细胞衰老。PM2.5在肺内的累积是否通过诱导AT2细胞的衰老，损害其修复肺泡上皮黏膜的能力尚未见报道。既往研究多采用肺上皮细胞系或气管滴注动物模型。细胞系失去了原代细胞的许多特性，因此基于细胞系的研究成果不能精准反映体内真实情况。气管滴注动物模型往往存在PM2.5吸入肺内分布不均匀，且造成局部剂量太大，也偏离真实世界。为模拟真实场景，本课题建立大气直接采集PM2.5经浓缩后全身暴露染毒小鼠模型。日均暴露剂量为1.1mg/(m3h)，连续造模32天，累积暴露剂量为33.6mg/(m3h)。病理学分析显示小鼠肺部巨噬细胞增加，AT2细胞增殖加快。降低暴露频率或停止暴露6个月后肺部炎性改变消失。我们应用单细胞RNA测序技术对PM2.5暴露吸入一个月的小鼠肺组织进行降维聚类分析，绘制单细胞网络图谱。利用肺部细胞亚群基因的表达特征发现，处在增殖状态的AT2细胞数量增多，形成单独细胞群落。结合STRING、KEGG以及小鼠膜表面受体-配体数据库分析细胞间通讯作用以及信号通路提示PM2.5对AT2细胞的影响并非取决于单一信号成分，PM2.5中的多种成分，不同信号通路串扰共同影响AT2细胞的功能，启动损伤后的修复机制。分离小鼠原代AT2细胞进行类器官培养，PM2.5浓度分别为0.01mg/mL、0.05mg/mL、0.1mg/mL和0.2mg/mL。体外0.01mg/mL浓度与体内实验小鼠暴露剂量接近，此浓度PM2.5刺激诱导AT2细胞增殖加快，而随着浓度增加，AT2增殖显著下降。电镜扫描显示，PM2.5体外刺激AT2细胞，24小时后约2%的AT2细胞吞噬了颗粒物。微流控技术分析发现，PM2.5体外刺激可减低细胞电阻抗，提示PM2.5浓度升高可诱导AT2细胞衰老，并加速死亡。类器官培养证实H2O2抑制小鼠AT2细胞的增殖功能。本研究揭示PM2.5—活性氧自由基—肺泡干细胞衰老—肺泡上皮损伤的关系机制，阐明大气雾霾细颗粒物诱发肺损伤并损伤后修复的生物学机制。