SURFACTANT PROTEINS AND TYPE II CELL DIFFERENTIATION

表面活性蛋白和 II 型细胞分化

• 批准号: 6655311
• 负责人: PHILIP L. BALLARD
• 金额: $ 24.35万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6655311
• 关键词: bronchopulmonary dysplasia; cell component structure /function; cell differentiation; cellular pathology; embryo /fetus tissue /cell culture; fluorescence microscopy; granule; growth /development; human tissue; immunoelectron microscopy; intracellular transport; laboratory mouse; lipid transport; lung development; membrane activity; membrane lipids; nitric oxide; protein biosynthesis; protein structure function; protein transport; pulmonary surfactants; respiratory epithelium; surface property; tissue /cell culture; western blottings

(Applicant's Abstract) A deficiency of pulmonary surfactant at birth is a major contributing cause of lung injury and long-term lung disease such as bronchopulmonary dysplasia (BPD). The severe respiratory distress associated with inherited deficiency of surfactant protein-B (SP-B), in both mice pups and infants born at term, indicates a key role for the hydrophobic SPs in differentiation of type II cells. In the absence of SP-B there is a failure of normal lamellar body genesis as well as incomplete processing of SP-C. Recently, isolated deficiency of SP-C has been described in infants with interstitial lung disease. Respiratory distress also occurs in newborn term BWB calves which lack mature SP-C and have reduced SP-B, and in rodents respiratory distress and acquired deficiency of SP-B/-C occurs with lung injury secondary to bleomycin or infection (P. carinii and endotoxin). Based on these and other findings, this project proposes that synthesis of SP-B, SP-C and lamellar bodies are closely linked and that relative levels of both SP-B and SP-C influence surfactant function. The objectives of this proposal are to characterize the biosynthetic pathway for human SP-C, determine the roles of SP-B and SP-C in lamellar body genesis, and investigate SP-B and SP-C in lung disease. Aim I will determine expression of mature SP-C during type II cell differentiation in vivo and in vitro in relationship to production of SP-B and lamellar bodies and also define targeting domains and cleavage events in SP-C processing. The studies will utilize antibody to mature SP-C and a recently developed culture system for hormonally induced type II cell differentiation in vitro. Aim II will investigate the role and interactions of SP-B and SP-C in lamellar body genesis and trafficking of surfactant components using cell culture models of SP deficiency. The studies will examine the hypothesis that expression of mature SP-B is required for both lamellar body formation and final processing of SP-C intermediates. Experiments will be carried out in the cultured type II cell model and SP-B or -C gene expression will be selectively inhibited using adenovirus expressing antisense mRNAs. Processing and intracellular trafficking of each SP will be studied using epitope specific antibodies, pulse/chase labeling, and tagged recombinant proteins. In addition, processing and effects of alternatively spliced SP-B and mutated SP-C will be determined. Aim III will investigate expression of SP-B and SP-C in surfactant from infants with lung disease and after treatment with inhaled nitric oxide. It is hypothesized that a deficiency of SP-B and/or SP-C occurs in infants with severe BPD, and that this process is modulated by anti-inflammatory effects of nitric oxide. In addition, the developmental pattern for alternative SP-B splicing in human lung and relationship of splicing variants to SP-B levels and newborn lung disease will be determined. The proposed studies will utilize both the Tissue Culture and Clinical Cores and involve collaboration with Projects 6, 4 and 7. The new information will provide further understanding of the role of the hydrophobic surfactant proteins in lung development and newborn lung diseases.

（申请人摘要）出生时肺表面活性物质缺乏是一种 肺损伤和长期肺部疾病的主要原因，例如 支气管肺发育不良（BPD）。相关的严重呼吸窘迫 两只幼鼠均患有表面活性蛋白 B (SP-B) 遗传缺陷 和足月出生的婴儿，表明疏水性 SP 在 II型细胞的分化。如果没有 SP-B，就会出现故障 正常层状体发生以及 SP-C 加工不完整。 最近，在患有以下疾病的婴儿中发现了孤立的 SP-C 缺乏症： 间质性肺疾病。新生儿足月也会出现呼吸窘迫 缺乏成熟 SP-C 且 SP-B 减少的 BWB 小牛，以及啮齿类动物 肺发生呼吸窘迫和获得性 SP-B/-C 缺乏 博莱霉素或感染（卡氏疟原虫和内毒素）继发的损伤。基于 根据这些和其他发现，该项目建议合成 SP-B、SP-C 和层状体紧密相连，并且 SP-B 的相对水平 SP-C影响表面活性剂功能。该提案的目标是 表征人类 SP-C 的生物合成途径，确定其作用 SP-B 和 SP-C 在板层体发生中的作用，并研究 SP-B 和 SP-C 肺部疾病。目标 I 将确定 II 型期间成熟 SP-C 的表达 体内和体外细胞分化与 SP-B 产生的关系 和层状体，还定义了靶向域和裂解事件 SP-C加工。这些研究将利用成熟 SP-C 的抗体和 最近开发的激素诱导 II 型细胞培养系统 体外分化。目标 II 将调查角色和相互作用 SP-B 和 SP-C 在层状体生成和表面活性剂运输中的作用 使用 SP 缺乏细胞培养模型的成分。这些研究将 检查以下假设：成熟 SP-B 的表达对于两者都是必需的 SP-C中间体的层状体形成和最终加工。 实验将在培养的II型细胞模型和SP-B或 -C基因表达将通过腺病毒表达被选择性抑制 反义 mRNA。每个 SP 的加工和细胞内运输将 使用表位特异性抗体、脉冲/追踪标记和标记进行研究 重组蛋白。另外，替代的处理和效果 将确定剪接的SP-B和突变的SP-C。目标 III 将进行调查 肺病婴儿表面活性剂中 SP-B 和 SP-C 的表达 吸入一氧化氮治疗后。假设一个 患有严重 BPD 的婴儿会出现 SP-B 和/或 SP-C 缺乏，并且 这一过程受到一氧化氮的抗炎作用的调节。在 此外，人类选择性 SP-B 剪接的发育模式 肺以及剪接变异与 SP-B 水平和新生儿肺的关系 疾病就会被确定。拟议的研究将利用组织 文化和临床核心并涉及与项目 6、4 和 7. 新信息将使人们进一步了解 肺发育和新生肺中的疏水性表面活性蛋白 疾病。