Novel reagents for alveolar type I and type II cells

描述（由申请人提供）：本赠款申请涉及广泛的挑战领域（直接摘自NHLBI对RFA-OD-09-003的描述：NHLBI挑战赠款。我们已在此挑战补助金中与此申请相关的部分下划了下划线。）“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。用于研究肺细胞生物学和疾病进展的试剂缺乏。实例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性敲除转基因动物的启动子，以及识别细胞表面标志物并可用于FACS分选气道中不同细胞谱系的抗体。这些标记物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部细胞变化也很重要。它们还可以用作肺部疾病进展的替代物和用于解剖肺细胞类型的细胞异质性/功能。“肺泡上皮细胞覆盖了肺内表面积的99%以上，由两种类型的细胞组成，I型和II型细胞，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型细胞的非常薄（约50 nm）的细胞质延伸覆盖将上皮与间质和血管隔室分开的基底膜。II型细胞是较小的立方形细胞（直径为100 mm）。~10 <$m），形态上以称为板层体的分泌颗粒为特征。虽然I型细胞的功能不如II型细胞的功能了解，但I型细胞被认为在肺中起重要作用，因为它们覆盖肺的98%以上的内表面积，在空气和血液隔室之间提供了对有效气体交换至关重要的狭窄解剖屏障。由于I型细胞具有极高的水渗透性和转运离子的能力，I型细胞可能在肺液体稳态中起重要作用。II型细胞覆盖肺泡表面的其余部分，合成、分泌和再循环表面活性剂组分，具有转运离子、合成免疫效应分子和在肺泡上皮损伤后充当祖细胞的能力。我们对这一挑战的回应主要集中在两种类型的试剂的开发上：1）用于产生对肺中的I型或II型细胞特异性的条件性敲除的探针，其也将赋予对这些细胞进行FACS分选的能力;和2）表征对人I型或II型细胞特异性识别的蛋白质抗原，用于FACS分选人肺泡上皮细胞和用于开发更可靠的检测方法来研究肺损伤和其他损害肺泡上皮的疾病的进展。有两个具体目标：1）产生转基因动物，所述转基因动物可用于在肺泡上皮I型或II型细胞中特异性的条件性敲除和用于相同细胞的FACS分选。2)开发鉴定I型或II型细胞特异性顶端质膜的改良试剂，其适用于人肺泡上皮细胞的FACS分选，并用作评价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，以生产用于肺泡上皮细胞生物学和人类肺部疾病研究的新型试剂。 公共卫生相关性：我们对这一挑战的回应主要集中在开发两种类型的试剂：1）用于产生对肺中的I型或II型细胞特异性的条件性敲除的探针，其将允许同时进行细胞的FACS分选;和2）表征由对人I型或II型细胞特异性的抗体识别的蛋白质抗原，用于FACS分选人肺泡上皮细胞和用于开发更可靠的检测方法来研究肺损伤和其他损害肺泡上皮的疾病的进展。拟议的研究是我们实验室以前工作的自然延伸，但目前没有资金。

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.