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.

（申请人的摘要）出生时肺表面活性剂的缺乏是 肺损伤和长期肺部疾病的主要原因 支气管肺发育不良（BPD）。严重的呼吸窘迫 两只小鼠幼崽的表面活性剂蛋白-B（SP-B）的遗传缺乏 和学期出生的婴儿表示疏水SP的关键作用 II型细胞的分化。在没有SP-B的情况下，有失败 正常的层状体发生以及SP-C的不完整处理。 最近，在婴儿的婴儿中描述了SP-C的孤立缺陷 间质性肺部疾病。呼吸窘迫也发生在新生期间 BWB犊牛缺乏成熟的SP-C并且减少了SP-B，在啮齿动物中 肺部呼吸窘迫和获得的SP-B/-C的缺乏发生在肺中 继发于博来霉素或感染的损伤（Carinii和内毒素）。基于 在这些和其他发现，该项目提出了SP-B，SP-C的合成 层状体紧密相连，两个SP-B的相对水平 SP-C影响表面活性剂功能。该提议的目标是 要表征人类SP-C的生物合成途径，请确定角色 层状体发生的SP-B和SP-C，并研究SP-B和SP-C 肺部病。目的我将在II型期间确定成熟的SP-C的表达 细胞分化体内和体外与SP-B产生的关系 和层状体，还定义了目标域和裂解事件 SP-C处理。研究将利用对成熟SP-C和A的抗体 最近开发的荷尔蒙诱导II型细胞的培养系统 体外分化。 AIM II将调查角色和互动 Sp-B和Sp-C的层状体发生和表面活性剂的贩运 使用SP缺乏的细胞培养模型的组件。研究会 检查两个假设，即两者都需要成熟的SP-B表达 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基因表达将使用表达腺病毒选择性抑制 反义mRNA。每个SP的处理和细胞内贩运将是 使用表位特异性抗体，脉冲/追逐标签进行了研究和标记 重组蛋白。另外，处理和效果替代 将确定剪接的SP-B和突变的SP-C。 AIM III将调查 来自肺部疾病的婴儿的表面活性剂中SP-B和SP-C的表达 用吸入一氧化氮处理后。假设一个 SP-B和/或SP-C的缺乏发生在严重BPD的婴儿中，并且 一氧化氮的抗炎作用调节了此过程。在 此外，人类替代SP-B剪接的发育模式 肺和剪接变体与SP-B水平和新生儿肺的关系 将确定疾病。拟议的研究将利用两组 文化和临床核心，并与项目6、4和 7。新信息将进一步了解 肺发育中的疏水表面活性剂蛋白和新生儿肺 疾病。