Regenerative Role of Integrin Beta4pos p63pos Distal Lung Stem/Progenitor Cells

描述(由申请人提供)：远端肺上皮具有关键的生物学功能，包括气体交换和提供屏障，以防止有害的空气传播物质进入人体。它也是许多遗传性和获得性疾病的目标，超过10%的美国人口患有慢性肺部疾病。尽管这一组织很重要，但对于维持和修复这一器官的重要细胞类型仍存在相当大的分歧。在气管中，基底细胞是关键的前体细胞类型，而Clara细胞被认为是远端呼吸道中更重要的前体细胞，特别是在气管外缺乏基底细胞的小鼠中。肺泡II型细胞 长期以来一直被确定为肺泡的重要祖先种群，这一范式在最近的研究中反复得到证实。此外，几个研究小组已经发现，没有成熟谱系标记的细胞是肺泡细胞类型的潜在祖细胞。特别是，肺远端整合素4表达的细胞被认为参与了博莱霉素诱导的肺损伤后的肺泡修复，而细胞角蛋白5(Krt5)/p63的表达细胞参与了流感感染后的肺泡再生。我们未发表的工作表明，博莱霉素损伤后也出现了丰富的Krt5pos细胞，在两种损伤模型中，这些细胞都强烈表达β4。从未损伤的肺组织中分离的新鲜4+细胞进一步鉴定表明，这些细胞表达p63，但不表达Krt5。然而，在体外扩增时，Krt5表达上调，表明这些细胞倾向于采用基础细胞样表型。这项建议的主要目的是确定这些远端p63+和4+实际上是否是损伤后扩大的Krt5+细胞的起源细胞，并确定这些细胞在多大程度上负责肺泡修复/再生。我们将针对这一假说提出以下具体目标：1)确定4+p63+细胞对肺损伤的活体激活反应。2)检测p63的调节表达是否对肺远端4+Krt5+细胞获得Krt5+再生表型起关键作用。几种转基因小鼠品系、肺切片培养成像和体外分子生物学技术将被用来实验地解决这些目标。这些研究将有助于更好地了解肺上皮细胞对损伤的反应，并将为如何利用/引导肺祖细胞用于治疗目的提供见解。

DESCRIPTION (provided by applicant): The epithelium of the distal lung performs critical biological functions including gas exchange and providing a barrier to prevent access of deleterious airborne agents into the body. It is also the target of numerous inherited and acquired diseases, represented by over 10% of the US population suffering from chronic lung disease. Despite the importance of this tissue, there is considerable disagreement as to the cell types important for the maintenance and repair of this organ. In the trachea, basal cells are the key progenitor cell type, while clara cells are thought to be more important progenitors in distal airways, especially in mice which lack basal cells outside the trachea. Alveolar type II cells have been long established as an important progenitor population for alveoli, a paradigm repeatedly confirmed in recent studies. In addition, several groups have identified cells that bear no mature lineage markers as potential progenitors for alveolar cell types. Specifically, distal lung integri ¿4-expressing cells are proposed to contribute to alveolar repair after bleomycin-induced injury, while cytokeratin 5(Krt5)/p63-expressing cells have been implicated in alveolar regeneration after influenza infection. Our unpublished work demonstrates that abundant Krt5pos cells also appear after bleomycin injury, and in both injury models these cells strongly express ¿4. Further characterization of freshly isolated ¿4+ cells from uninjured lungs revealed that these cells express p63, but not Krt5. However, upon expansion ex vivo, Krt5 is upregulated, demonstrating the propensity of these cells to adopt a basal cell-like phenotype. The major goal of this proposal is to determine whether these distal p63+ ¿4+ are in fact the cell-of-origin for expanded post-injury Krt5+ cells and to define the extent to which these cells are responsible for alveolar repair / regeneration. We will address this hypothesis with the following Specific Aims: 1) Determine the in vivo activation response of ¿4+ p63+ cells to lung injury. 2) Test whether regulated expression of p63 is critical to the acquisition of a Krt5+ regenerative phenotype by distal lung ¿4+Krt5+ cells. Several strains of transgenic mice, lung slice culture imaging, and in vitro molecular biology techniques will be used to address these aims experimentally. These studies will result in a better understanding of how lung epithelium responds to injury and will provide insights as to how lung progenitors might be exploited / directed towards therapeutic purposes.