FUNCTIONS OF PULMONARY ALVEOLAR TYPE II CELLS IN VITRO

II 型肺泡细胞的体外功能

• 批准号: 2216535
• 负责人: ROBERT James MASON
• 金额: $ 22.48万
• 依托单位: NATIONAL JEWISH HEALTH
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-01-01 至 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2216535
• 关键词: alveolar macrophages; autoradiography; cell growth regulation; diagnostic respiratory lavage; enzyme linked immunosorbent assay; gene expression; growth factor; iodine; laboratory rat; lung alveolus; northern blottings; phospholipids; pulmonary surfactants; radionuclides; radiotracer; receptor; receptor binding; secretion; tissue /cell culture

Alveolar type II cells are critical to the function of normal lung. These cells synthesize and secrete pulmonary surface active material and are the stem cells for the alveolar epithelium. In this proposal we will use rat type II cells to investigate the synthesis and secretion of surface active material and the proliferation of type II cells in vitro. Pulmonary surface active material is an heterogeneous mixture of phospholipids and the surfactant associated proteins (SP-A, SP-B, and SP-C). Surfactant protein A inhibits the secretion of phosphatidylcholine in vitro and stimulates the uptake of liposomal lipid into type II cells. We have recently shown that SP-A binds to type II cells with high affinity and specificity. To date, the receptor has not been isolated. However, digestion of type II cell plasma membranes with endoglycoceramidase indicates that the high affinity receptor is a glycosphingolipid. Our first specific aim is to isolate and characterize the type II cell receptor for SP-A. We will determine if the receptor required for inhibition of secretion is the same as the receptor required for the stimulation of uptake of liposomal lipid by SP-A. The second aim is to determine how phospholipids and SP-A inhibit secretion in vitro. We will elucidate the structural basis for phospholipid mediated regulation of secretion and determine at which point in the secretory cascade phospholipids act. Since SP-A inhibits multiple agonists, we believe that its effects are distal to the initial receptor activation and generation of second messengers. Our hypothesis is that small molecular weight guanine nucleotide binding proteins or the subplasma membrane cytoskeleton are altered. We recently developed a primary culture system for maintaining differentiation of type II cells in vitro. In our third specific aim we will use this culture system to study longer term regulation of surfactant synthesis and secretion in vitro. We will determine whether or not the protein and the lipids of surfactant are secreted together and the regulation of secretion in the presence of extracellular surfactant. Type II cells proliferate to maintain the alveolar epithelium and can be stimulated to proliferate in vitro. Two sources of growth factors that stimulate proliferation in vitro and are potentially important in vivo are bronchoalveolar lavage fluid and media conditioned by macrophages. Our fourth specific aim is to identify and purify growth factors from these sources and establish whether or not they are known or novel growth factors. Our fifth specific aim is to demonstrate that the type II cells which proliferate in vitro can re- express the type II cell phenotype under appropriate culture conditions in vitro.

牙槽II型细胞对于正常肺的功能至关重要。 这些 细胞合成并分泌肺表面活性材料，是 牙槽上皮的干细胞。 在此提案中，我们将使用老鼠 II型细胞研究表面活性的合成和分泌 物质和II型细胞体外的增殖。 肺 表面活性材料是磷脂的异质混合物， 表面活性剂相关蛋白（SP-A，SP-B和SP-C）。 表面活性剂 蛋白A抑制体外磷脂酰胆碱的分泌 刺激脂质体脂质对II型细胞的摄取。 我们有 最近表明，SP-A与具有高亲和力的II型细胞结合， 特异性。 迄今为止，尚未隔离受体。 然而， 用内糖果酰胺酶消化II型细胞质膜 表明高亲和力受体是糖磷脂。 我们的 第一个具体目的是隔离和表征II型细胞受体 对于sp-a。 我们将确定是否需要抑制受体 分泌与刺激所需的受体相同 SP-A摄入脂质体脂质。 第二个目的是确定如何 磷脂和SP-A体外抑制分泌。 我们将阐明 磷脂介导的分泌和分泌调节的结构基础 确定《分泌级联磷脂法》中的哪个点。 自从 SP-A抑制多种激动剂，我们认为它的作用是 初始受体激活和第二使者的产生。 我们的 假设是小分子量鸟嘌呤核苷酸结合 蛋白质或亚皮膜膜细胞骨架发生了变化。 我们最近 开发了一种主要的培养系统，用于维持类型的区分 II细胞体外。 在我们的第三个特定目标中，我们将使用这种文化 研究表面活性剂合成的长期调节和 体外分泌。 我们将确定是否蛋白质和 表面活性剂的脂质被分泌在一起，分泌的调节 在细胞外表面活性剂的情况下。 II型细胞增殖至 维持牙槽上皮，可以刺激以增殖 体外。 生长因子的两个来源，刺激体外增殖 并且在体内可能很重要的是支气管肺泡灌洗液和 媒介以巨噬细胞为条件。 我们的第四个具体目的是确定 并从这些来源净化增长因素，并确定是否 它们是已知的或新颖的增长因素。 我们的第五个具体目标是 证明体外增殖的II型细胞可以重复 在适当的培养条件下表达II型细胞表型 体外。