PERMEABILITY OF THE LUNG TO WATER SOLUBLE SOLUTES

肺对水溶性溶质的渗透性

• 批准号: 6182904
• 负责人: EVELINE ELSA SCHNEEBERGER
• 金额: $ 33.53万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-12-20 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6182904
• 关键词: enzyme inhibitors; freeze etching; histology; human subject; immunocytochemistry; intracellular transport; membrane biogenesis; membrane lipids; membrane permeability; membrane structure; membrane transport proteins; phlebotomy; phospholipase C; protein kinase C; protein transport; radiotracer; respiratory epithelium; tight junctions; tissue /cell culture; transfection

The long term objective of the proposed research is to analyze the mechanisms by which endothelial and epithelial cells regulate the permeability properties of the pulmonary air-blood barrier. To accomplish this two model systems are employed. 1. The plasma protein-free fluorocarbon exchange transfused rat and 2. epithelial cells in culture. Biochemical and electrophysiological data are correlated with observations made by ultrastructural immunocytochemistry, morphometry and freeze fracture. The specific aims include studies utilizing the fluorocarbon exchange transfused rat, to determine the ability of immunoglobulin G (IgG), alpha1- acid glycoprotein (alpha1-AGP), fibrinogen (FAN), plasma fibronectin (FN) and dibutyryl cAMP, alone and in combination, to augment the action of albumin in preventing the increased macromolecular transport and edema that occurs in a protein-free preparation. The localization of administered test proteins on the pulmonary endothelium will be determined immunocytochemically, and correlated with morphometric measurements of ferritin movement, a measure of macromolecular transport. The distribution of ZO-1, a TJ associated protein, is mapped immunocytochemically in microvascular endothelial cells under conditions of normal and augmented fluid filtration. The plasmid pZipSVtsA58, a construct that confers temperature sensitive growth potential to transfected epithelial cells at 33oC, but allows expression of differentiated functions at 39oC, is used to immortalize alveolar type II cells. Clones that develop a transepithelial resistance >100 omega cm2 are selected for study of the regulation and composition of TJs. Novel strategies using biotinylating agents and a heterobifunctional crosslinking agent are employed to identify and characterize TJ protein(s) to which monoclonal antibodies will be raised. The role of membrane lipids in the regulation and function of TJs will be examined in cultured epithelial cells which, when made cholesterol deficient by administration of Lovastatin, develop steady state transepithelial resistances two to three-fold greater than controls. Results of the proposed research will: i. Demonstrate the key role played by specific plasma proteins, in addition to albumin, in regulating endothelial permeability. ii. Identify TJ proteins and provide reagents with which to study their structure using tools of molecular biology and iii. aid in resolving controversy concerning the role of membrane lipids in TJs. A better understanding of the nature of this important structure will provide a rational basis for the management of pulmonary edema.

拟议研究的长期目标是分析 内皮细胞和上皮细胞调节血管内皮细胞生长的机制 肺气血屏障的通透性。要完成 采用了这两个模型系统。1.无蛋白血浆 氟碳交换输注大鼠和2.培养中的上皮细胞。 生化和电生理数据与观察值相关 经超微结构免疫细胞化学、形态计量学和冷冻 骨折。 具体目标包括利用氟碳交换的研究。 输注大鼠，测定免疫球蛋白G(Ig G)、α1- 酸性糖蛋白(α1-AGP)、纤维蛋白原(FAN)、血浆纤维连接蛋白(FN) 和二丁酰cAMP，单独和组合，以增强 白蛋白在预防大分子转运增加和脑水肿中的作用 在不含蛋白质的制剂中发生。被管理人的本土化 将测定肺内皮细胞上的测试蛋白 免疫细胞化学，并与形态计量学测量相关 铁蛋白运动，一种衡量大分子运输的指标。分布情况 ZO-1是一种TJ相关蛋白，免疫细胞化学定位于 正常和增强状态下的微血管内皮细胞 液体过滤。PZipSVtsA58，一种赋予 转基因上皮细胞对温度敏感的生长潜能 33oC，但允许在39oC下表达不同的功能，用于 使肺泡II型细胞永生化。发展成跨上皮细胞的克隆 选择100 omega cm2用于研究调节和 TJ的构成。使用生物素化试剂和 使用异双官能团交联剂来鉴定和 确定TJ蛋白(S)的特性，并将其提升至单抗。 膜脂在TJs的调节和功能中的作用将是 在培养的上皮细胞中进行检查，当产生胆固醇时 洛伐他汀治疗不足，病情稳定 跨皮细胞阻力比对照组大两到三倍。 拟议研究的结果将：i.论证所发挥的关键作用 通过特定的血浆蛋白，除了白蛋白，在调节 内皮通透性。二、鉴定TJ蛋白并提供试剂 用分子生物学和分子生物学的工具研究它们的结构 三、帮助解决关于膜脂作用的争议 穿着TJ。更好地了解这一重要结构的性质 将为肺水肿的治疗提供合理依据。