衰老的发生会对生命个体的健康产生系统性的影响。而衰老对于不同组织或细胞也具有显著的差异。随着衰老的发生，肺组织的生理结构和功能会发生不可逆的改变，并伴随多种肺疾病的发生。其中肺泡上皮二型细胞作为主要的肺泡上皮干细胞，在维持肺泡的生理功能和肺泡损伤后修复中都具有重要作用。这些功能也会受到衰老的影响发生显著的改变。大量研究表明肺泡上皮二型细胞功能的紊乱会直接影响肺泡再生，甚至导致多种肺疾病的发生。但是目前对于在肺泡再生过程中衰老对肺泡上皮二型细胞的影响及其调控机制仍然缺乏深入的研究。本论文通过结合小鼠遗传学、单细胞测序等技术方法，研究了衰老对肺泡上皮二型细胞功能的影响的细胞和分子机制。

通过对比分析了年轻和年老小鼠在博来霉素诱导的肺泡再生过程，我们发现年老小鼠的肺泡再生能力发生了显著降低。进一步的谱系示踪实验结果表明，年老小鼠的肺泡上皮二型细胞的增殖能力并没有改变，但分化成肺泡上皮一型细胞的能力却显著降低，从而使得这些细胞在损伤区域呈聚集状分布。

为了研究其中的细胞和分子机制，我们通过单细胞测序从转录组水平研究分析了损伤后的年轻和年老小鼠的肺泡上皮二型细胞。我们发现这些肺泡上皮二型细胞可以分为三个不同的细胞亚群。其中有一个细胞亚群的比例在损伤后的年老小鼠中显著增加。由此，我们推测这群细胞与年老小鼠肺泡再生能力的降低有关系。所以，我们进一步比较了年轻和年老小鼠中的这群细胞的差异表达基因，发现年老小鼠肺泡上皮二型细胞中腺苷酸活化蛋白激酶（AMP-activated protein kinase，AMPK）信号通路相关基因表达显著低于年轻小鼠。

为了验证AMPK信号通路的降低是否是导致年老小鼠肺泡上皮二型细胞分化能力降低的原因，我们首先检测了损伤后年轻和年老小鼠的肺组织中AMPK信号通路的活性。实验结果表明，在损伤后，年轻小鼠的肺泡上皮二型细胞内的AMPK会被明显激活，而年老小鼠的则不能被激活。然后，我们通过小鼠遗传学的方法将Stk11从肺泡上皮二型细胞中特异性敲除来验证AMPK信号通路的功能。实验结果表明，在肺泡损伤后，Stk11的敲除会显著降低肺泡上皮二型细胞内AMPK信号通路的活性，并且显著抑制细胞分化成肺泡上皮一型细胞的能力。

最后，我们通过使用小分子化合物AICAR激活年老小鼠的肺泡上皮二型细胞中的AMPK信号通路来进一步验证AMPK在年老小鼠中的功能。在AICAR药物处理后，不论是在体外肺泡小体培养实验还是体内实验中，我们都发现激活AMPK能够显著促进肺泡上皮二型细胞的分化。

综上所述，我们发现Stk11-AMPK信号通路对于肺泡上皮二型细胞的分化是必需的，而由于衰老引起细胞内AMPK信号通路的降低，导致了肺泡上皮二型细胞不能分化成肺泡上皮一型细胞。通过以上研究,我们阐述和证明了衰老对肺泡干细胞功能的影响及其调控机制。这些结果为我们进一步研究衰老与肺脏再生和疾病发生之间的关系提供了新的思路，也为相关疾病的治疗提供了可能的靶点。

Aging directly affects the health of living organisms. With the onset of aging, the physiological structure and function of lung will undergo the irreversibly changes, which strongly correlated with the occurrence of multiple lung diseases. Alveolar epithelial type 2 cells function as the alveolar stem cells and play the key roles in maintaining the alveolar physiological function and pathological injury repair. The functions of alveolar epithelial type 2 cells are also significantly altered by aging. Previous studies have shown that the dysfunction of alveolar epithelial type 2 cells would lead to multiple aging related-lung diseases. However, little is known about the mechanisms of aging on alveolar epithelial type 2 cells during alveolar regeneration. In this thesis, we study the cellular and molecular mechanisms on how aging affects alveolar regeneration by combining mouse genetics and single-cell RNA sequencing technology. 
By comparing alveolar regeneration processes of young and aged mice, we found that the alveolar regeneration capacity of aged mice was significantly decreased. In addition, we observed alveolar epithelial type 2 cells of aged mouse show impaired differentiation and kept clustered in alveolar injury area.
To futher study the cellular and molecular mechanisms, we performed single-cell RNA sequencing and then analyzed the transcriptomic changes of young and aged alveolar epithelial type 2 cells. Based on the features of transcriptome, we found alveolar epithelial type 2 cells contain three different cell subpopulations. A subpopulation that enriched the genes related with alveolar regeneration was significantly increased in aged mice after alveolar injury. By further comparing the differentially expressed genes of this subpopulation between young and aged mice, we found that genes related with AMPK signaling pathway were significantly decreased in the aged alveolar epithelial type 2 cells.
To investigate whether decreased AMPK signaling results in impaired differentiation of aged mice，we firstly analyzed AMPK activities in young and aged mice after lung injury.The results show that AMPK was only activated in young alveolar epithelia type 2 cells,but not in aged alveolar epithelia type 2 cells. Next, we specifically knocked out Stk11 from alveolar epithelial type 2 cells to validate AMPK functions in alveolar regeneration. The results show that knocking out Stk11 would significantly decrease AMPK signaling activity, and impaired alveolar epithelial type 2 cell differentiation.
Finally, we used AICAR treatment to activate the AMPK signaling in alveolar epithelial type 2 cells in aged mice to demonstrate AMPK functions in aged mice. After AICAR treatment, we found that the differentiation of alveolar epithelial type 2 cells in aged mice was significantly improved both in vitro alveolar organoid culture and in vivo experiments.
In summary, we demonstrated that the Stk11-AMPK signaling axis is required for the differentiation of alveolar epithelial type 2 cells and impaired differentiation of alveolar epithelial type 2 cells in aged mice is caused by decreased AMPK signaling. These findings provide us new insights for understanding the cellular and molecular mechanisms in lung regeneration and diseases.