Laminin-5 in Lung Develooment and Disease

Laminin-5 在肺发育和疾病中的作用

• 批准号: 6823501
• 负责人: ROBERT M SENIOR
• 金额: $ 22.5万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6823501
• 关键词: Pseudomonas aeruginosa; basement membrane; cell migration; emphysema; extracellular matrix; gene expression; gene targeting; genetically modified animals; immunofluorescence technique; inflammation; laboratory mouse; laminin; lung; lung development; lung injury; metalloendopeptidases; neutrophil; pulmonary fibrosis /granuloma; regeneration; respiratory epithelium; smoke inhalation; transmission electron microscopy; western blottings

Basement membranes (BMs) are necessary for lung development and restoration of normal alveolar architecture when the lung has sustained damage. However, little is known about alveolar BMs in the context of alveolar damage and repair. Laminins are integral BM glycoproteins. Laminin-5 (Ln-5), comprised of laminin chains alpha3, beta3, and gamma2 affects epithelial cell structure and function generally, and is present in alveolar BMs, but its roles specifically in lung development and alveolar damage are unknown. In Aim I we will study the role of Ln-5 in lung development and alveolar damage using mice deficient in Ln-5 as a result of targeting the laminin gamma2 gene. However, mice with a global knockout of the laminin gamma2 gene die <5 days after birth with mucosal and cutaneous sloughing. Because of this we will generate lung-specific, inducible laminin gamma 2-deficient mice. These mice will be used to examine the effect of Ln-5 deficiency on the later stages of lung development and the response to various models of alveolar damage, including Pseudomonas aeruginosa-induced alveolitis, bleomycin-induced pulmonary fibrosis, and cigarette smoke-induced emphysema. Their lungs will be evaluated for structure, expression of extracellular matrix genes and inflammatory cell recruitment. In Aim II we will assess the effects of Ln-5 on alveolar type epithelial cell (AEC) migration. These studies will involve AECs isolated from wild type mice and mice with the lung- specific, inducible knockout of laminin 72. In Aim III, using in vivo and in vitro models like those described in Aims I and II, we will study mice with deficiencies of matrix metalloproteinases (MMPs) -2 or -14 to determine the roles of these MMPs on responses to alveolar injury and AEC cell migration. MMPs -2 and -14 are of particular interest because they are known to affect epithelial cell migration via effects upon Ln-5, but their role in AEC functions has not been determined. Because neutrophils are often prominent in alveolar injury we will also explore the effects of neutrophil proteinases upon Ln-5 and its effects on AEC cell functions. The studies proposed in this grant application will contribute new information about alveolar BMs, an important structure in serious, common lung disorders, including the acute respiratory distress syndrome, pulmonary fibrosis, and emphysema.

当肺部受到持续损伤时，基底膜（BM）对于肺部发育和正常肺泡结构的恢复是必需的。然而，人们对肺泡损伤和修复中的肺泡 BM 知之甚少。层粘连蛋白是完整的 BM 糖蛋白。层粘连蛋白-5 (Ln-5) 由层粘连蛋白链 α3、β3 和 γ2 组成，通常影响上皮细胞结构和功能，并且存在于肺泡 BM 中，但其在肺发育和肺泡损伤中的具体作用尚不清楚。为了目标，我们将 使用因靶向层粘连蛋白 gamma2 基因而缺乏 Ln-5 的小鼠研究 Ln-5 在肺发育和肺泡损伤中的作用。然而，层粘连蛋白γ2基因全面敲除的小鼠在出生后5天内就会死亡，并伴有粘膜和皮肤脱落。因此，我们将产生肺特异性、可诱导层粘连蛋白 γ 2 缺陷的小鼠。这些小鼠将用于检查 Ln-5 缺乏症对肺发育后期的影响以及对各种肺泡损伤模型的反应，包括铜绿假单胞菌诱导的肺泡炎、博莱霉素诱导的肺纤维化和香烟烟雾诱导的肺气肿。将评估他们肺部的结构、细胞外基质基因的表达和炎症细胞的募集。在目标 II 中，我们将评估 Ln-5 对肺泡型上皮细胞的影响 （AEC）迁移。这些研究将涉及从野生型小鼠和肺特异性、诱导性敲除层粘连蛋白 72 的小鼠中分离出的 AEC。在 Aim III 中，使用像 Aims I 和 II 中所述的体内和体外模型，我们将研究具有基质金属蛋白酶 (MMP) -2 或 -14 缺陷的小鼠，以确定这些 MMP 对肺泡损伤和 AEC 细胞反应的作用。 迁移。 MMP -2 和 -14 特别令人感兴趣，因为已知它们通过影响 Ln-5 来影响上皮细胞迁移，但它们在 AEC 功能中的作用尚未确定。由于中性粒细胞在肺泡损伤中通常很突出，我们还将探讨中性粒细胞蛋白酶对 Ln-5 的影响及其对 AEC 细胞功能的影响。本拨款申请中提出的研究将提供有关肺泡 BM 的新信息， 它是严重常见肺部疾病的重要结构，包括急性呼吸窘迫综合征、肺纤维化和肺气肿。