Novel reagents for alveolar type I and type II cells

描述（由申请人提供）：该资助申请涉及广泛的挑战领域（直接取自RFA-OD-09-003: NHLBI挑战资助的NHLBI描述）。我们已经强调了挑战奖助金中与此申请相关的部分。)06：使能技术，挑战主题：06- hl -109：生成用于研究肺细胞生物学和疾病进展的试剂。缺乏研究肺细胞生物学和疾病进展的试剂。例子包括识别特定细胞类型的抗体，仅在某些细胞类型中表达并可用于产生条件敲除转基因动物的启动子，以及识别细胞表面标记并可用于气道中不同细胞系的FACS分选的抗体。这些标记不仅对了解肺细胞类型的异质性很重要，而且对了解肺部疾病引起的肺细胞变化也很重要。它们也可以作为肺部疾病进展的替代品，用于解剖肺细胞类型的细胞异质性/功能。”肺泡上皮覆盖了肺内表面积的99%，由I型和II型细胞两种类型的细胞组成，这两种细胞都被认为是哺乳动物生命所必需的。I型细胞是非常大的鳞状细胞，计算直径为50-100 μ m；I型细胞的细胞质延伸非常薄（~50 nm），覆盖着将上皮细胞与间质室和血管室分开的基膜。II型细胞是较小的立方体细胞（直径~10¿m），其形态学特征是分泌颗粒，称为板层体。尽管I型细胞的功能比II型细胞的功能更不清楚，但I型细胞被认为在肺中起着重要作用，因为它们覆盖了肺内表面积的98%以上，在空气和血液间提供了一个狭窄的解剖屏障，对有效的气体交换至关重要。I型细胞具有极高的透水性和离子转运能力，可能在肺液体稳态中发挥重要作用。II型细胞覆盖肺泡表面的其余部分，合成、分泌和循环表面活性剂成分，具有转运离子、合成免疫效应分子的能力，并在肺泡上皮损伤后充当祖细胞。我们对这一挑战拨款的回应主要集中在两类试剂的开发上：1)用于产生肺中I型或II型细胞特异性条件敲除的探针，该探针也将赋予这些细胞FACS分选的能力；2)鉴定人类I型或II型细胞特异性识别的蛋白抗原，以便FACS分选人类肺泡上皮细胞，并开发更强大的检测方法来研究肺损伤和其他肺泡上皮损伤疾病的进展。有两个具体目的：1)产生转基因动物，可用于肺泡上皮I型或II型细胞的条件敲除，并用于相同细胞的FACS分选。2)开发可识别I型或II型细胞特异性顶质膜的改进试剂，这些试剂适用于人肺泡上皮细胞的FACS分选，并可作为评估各种肺损伤状态下疾病严重程度和进展的生物标志物。拟议的研究是我们实验室以前工作的自然延伸。TARP机制提供的资金将使我们能够在这两个重要的研究领域进行研究，生产用于肺泡上皮细胞生物学和人类肺部疾病研究的新试剂。

DESCRIPTION (provided by applicant): This grant application addresses broad challenge area (taken directly from the NHLBI description of RFA-OD-09-003: NHLBI Challenge Grants. We have underlined the portions of this Challenge Grant relevant to this application.) "06: Enabling technologies, Challenge Topic: 06-HL-109: Generate reagents for studying lung cell biology and disease progression. Reagents for studying lung cell biology and disease progression are lacking. Examples include antibodies that recognize specific cell types, promoters that are expressed only in certain cell types and can be used in the generation of conditional knockout transgenic animals, and antibodies that recognize cell surface markers and can be used for FACS sorting different cell lineages in the airway. Such markers would be important not only for understanding the heterogeneity of lung cell types but also for understanding cellular changes in the lung that emerge with lung disease. They may also be useful as surrogates for progression of lung disease and for dissecting cellular heterogeneity/function of lung cell types." The pulmonary alveolar epithelium, which covers >99% of the internal surface area of the lung, is comprised of two types of cells, type I and type II cells, both of which are believed to be essential for mammalian life. Type I cells are very large squamous cells with calculated diameters of 50-100 ¿m; the very thin (~50 nm) cytoplasmic extensions of the Type I cells cover the basement membrane separating the epithelial from the interstitial and vascular compartments. Type II cells are smaller, cuboidal cells (diam. ~10 ¿m) characterized morphologically by secretory granules called lamellar bodies. Although the functions of type I cells are less well understood than the functions of type II cells, type I cells are believed to play an important role in the lung because they cover more than 98% of the internal surface area of the lung, providing a narrow anatomic barrier between the air and blood compartments critical for efficient gas exchange. Type I cells may play an important role in lung liquid homeostasis by virtue of their properties of extremely high water permeability and capability of transporting ions. Type II cells, which cover the remainder of the alveolar surface, synthesize, secrete, and recycle surfactant components, have the capacity to transport ions, synthesize immune effector molecules, and act as progenitor cells following injury to the alveolar epithelium. Our response to this challenge grant focuses on the development of two types of reagents: 1) probes for the generation of conditional knockouts specific to either type I or type II cells in the lung that will also confer the ability to FACS sort these cells; and 2) characterization of protein antigens recognized by specific to human type I or type II cells for the purpose of FACS sorting human alveolar epithelial cells and for developing more robust assays to study progression of lung injury and other diseases that damage the alveolar epithelium. There are two specific aims: 1) To generate transgenic animals that can be used for conditional knockouts specifically in alveolar epithelial type I or type II cells and for FACS sorting of the same cells. 2) To develop improved reagents that identify type I or type II cell-specific apical plasma membranes that are suitable for FACS sorting of human alveolar epithelial cells and for use as biomarkers in evaluating disease severity and progression in various states of lung injury. The proposed studies are natural extensions of previous work in our laboratory. The funds available from the TARP mechanism would enable us to proceed in both of these important areas of investigation to produce novel reagents for the study of both lung alveolar epithelial cell biology and human lung disease. PUBLIC HEALTH RELEVANCE: Our response to this challenge grant focuses on the development of two types of reagents: 1) probes for the generation of conditional knockouts specific to either type I or type II cells in the lung that will permit simultaneous FACS sorting of cells; and 2) characterization of protein antigens recognized by antibodies specific to human type I or type II cells for the purpose of FACS sorting human alveolar epithelial cells and for developing more robust assays to study progression of lung injury and other diseases that damage the alveolar epithelium. The proposed studies are natural extensions of previous work in our laboratory but are currently unfunded.