Characterization of the Various Senescent Cells in the Aging Lungs

 描述(由申请人提供)：尽管有大量与年龄相关的生理参数变化的文件，但控制肺老化过程的机制尚未得到很好的研究。在已确定的与年龄相关的变化中，细胞外基质的改变(重塑)。这些变化导致老年肺对损伤和修复的反应发生改变。同样重要的是，肺部衰老细胞的数量随着年龄的增长而增加。然而，这些细胞引发肺部衰老的机制尚不确定。所有衰老的细胞都处于生长停滞状态，但继续分泌大量影响其微环境的蛋白质。似乎相互矛盾的数据表明，一方面，衰老的成纤维细胞有利于伤口的愈合，但另一方面，限制衰老的肺泡上皮细胞的数量有利于肺纤维化。因此，迫切需要表征存在于肺中的各种细胞的衰老轮廓 (主要是内皮细胞、上皮细胞和成纤维细胞)，以确定它们在衰老过程中的相对贡献。尽管在老年肺中检测到衰老细胞，但它们在与衰老相关的生理重塑中的作用尚不清楚，其分泌表型也仍不明确。在体内研究衰老在技术上具有挑战性；然而，我们最近获得了p16-3MR转基因系统，该系统能够在体内鉴定p16INK4a+细胞(衰老细胞)，并随后通过流式细胞仪分离它们，以及通过给药更昔洛韦来有条件地删除它们。我们建议使用这个小鼠模型来更明确地定义衰老细胞在肺老化中的作用；我们将在非人类灵长类动物衰老肺中验证我们的数据，这是一个人类衰老的翻译模型。我们假设，每种衰老细胞类型(肺泡上皮细胞、内皮细胞和成纤维细胞)都有一个与分泌相关的特征衰老曲线，以表明它们对衰老的相对贡献。针对这一假说提出了两个具体的目标：1)确定每种不同衰老细胞类型(肺泡上皮细胞、内皮细胞和成纤维细胞)与衰老相关的分泌谱，以确定它们在肺组织重塑中的作用。通过流式细胞术从非致命性照射的p16-3MR小鼠肺中分离出衰老和非衰老细胞，并将确定它们的转录图谱。差异表达的识别蛋白将在年轻和老年的非人类灵长类动物的肺中得到验证；以及2)确定衰老细胞在与年龄相关的肺重塑中的作用。利用p16-3MR转基因小鼠，我们将确定衰老细胞的存在是否通过在非致死剂量照射后删除p16INK4a+细胞来影响肺的重塑过程。通过该项目收集的数据将表征各种类型的衰老细胞的分泌情况，这些类型的细胞在血管重构中具有重要的功能作用。 不仅在肺中，而且在脑或肾脏等其他器官(衰老的内皮细胞和成纤维细胞)中也存在，并提供了关于机制的新信息。

 DESCRIPTION (provided by applicant): Despite ample documentation of age-related changes in physiologic parameters, mechanisms governing the process of aging of the lungs have not been well studied. Among the identified age-associated changes is the modification of the extracellular matrix (remodeling). These changes result in alteration of the aged lungs response to injury and repair. As importantly, the number of senescent cells in the lungs increases with age. However, the mechanisms by which these cells provoke aging of the lungs are uncertain. All senescent cells are in growth arrest but continue to secrete large amounts of proteins that affect their microenvironment. Seemingly contradictory data indicate that, on the one hand, senescent fibroblasts are beneficial in wound healing but, on the other hand, limiting the number of senescent alveolar epithelial cells is beneficial in pulmonary fibrosis. Thus, there is a critical need to characterize the senescence profile of the various cells present in the lungs (mainly endothelial, epithelial, and fibroblast cells) to determine their relative contribution to he aging process. Despite the detection of senescent cells in aged lungs, their role in physiological remodeling associated with aging is unknown and their secretory phenotype remains undefined. Studying senescence in vivo has been technically challenging; however, we have recently obtained the p16-3MR transgenic system that empowers in vivo identification of p16ink4A+ cells (senescent cells) and their subsequent isolation by flow cytometry as well as their conditional deletion via the administration of gancyclovir. We propose to use this mouse model to more definitely define the role of senescent cells in lung aging; we will validate our data in nonhuman primate aging lungs, a translational model to human aging. We hypothesize that each senescent cell type (alveolar epithelial, endothelial and fibroblasts) has a characteristic secretory associated senescence profile to indicate their relative contribution to aging. Two specific aims are proposed to address this hypothesis: 1) To identify the senescence-associated secretory profile of each different senescent cell type (alveolar epithelial and endothelial cells and fibroblasts) to determine their contribution to lung tissue remodeling. Senescent and non-senescent cells will be isolated from non-lethally irradiated p16-3MR mouse lungs by flow cytometry and their transcriptional profiling will be determined. The expression of differentially expressed identified proteins will be validated in young and aged nonhuman primate lungs; and 2) To determine the contribution of senescent cells in the age-associated remodeling of the lungs. Using the p16-3MR transgenic mouse, we will determine whether the presence of senescent cells affects the remodeling process in the lungs by deleting p16ink4A+ cells after non-lethal dose irradiation. The data gathered with this project will characterize the secretory profile of various senescent cell types that have an important functional role in remodeling in the lungs but also in other organs such as the brain or the kidney (senescent endothelial cells and fibroblasts) and provide new information as to mechanism.