SURFACTANT PROTEINS AND TYPE II CELL DIFFERENTIATION

(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，如 支气管肺发育不良(BPD)。与严重呼吸窘迫相关的 两只小鼠均存在遗传性表面活性蛋白B(SP-B)缺陷 和足月出生的婴儿，表明疏水性SPS在 II型细胞的分化。如果没有SP-B，则会出现故障 SP-C的正常板层小体发生以及不完全加工。 最近，孤立性SP-C缺乏症在婴儿中被描述为 间质性肺病。新生儿也会发生呼吸窘迫 缺乏成熟SP-C和降低SP-B的BWB小牛和啮齿类动物 肺部出现呼吸窘迫和获得性SP-B/-C缺乏症 继发于博莱霉素或感染(卡氏肺炎杆菌和内毒素)的损伤。基座 根据这些和其他发现，本项目建议合成SP-B、SP-C 和板层小体紧密相连，SP-B的相对水平 SP-C对表面活性物质的作用有影响。这项提议的目标是 为了表征人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，并探讨SP-B和SP-C在板层体发生中的作用 肺部疾病。目的检测成熟SP-C在II型中的表达。 体内外细胞分化与SP-B产生的关系 和板层小体，还定义了靶向结构域和切割事件 SP-C处理。这些研究将利用针对成熟SP-C和a的抗体。 新近建立的激素诱导的II型细胞培养体系 体外分化。AIM II将调查角色和相互作用 板层小体中SP-B和SP-C的含量表面活性物质的生成和迁移 采用细胞培养法建立SP缺乏症模型。这些研究将 检验成熟SP-B的表达对两者都是必需的假设 SP-C中间体的片体形成和最终加工。 实验将在培养的II型细胞模型和SP-B或 -C基因的表达将被选择性地抑制用腺病毒表达 反义mRNA。每个SP的处理和细胞内传输将是 使用表位特异性抗体、脉冲/追赶标记和标记进行研究 重组蛋白。此外，另一种方法的处理和效果 将确定拼接的SP-B和突变的SP-C。AIM III将进行调查 肺疾病患儿肺表面活性物质SP-B和SP-C的表达及意义 吸入一氧化氮治疗后。据推测，一个 重度BPD患儿存在SP-B和/或SP-C缺乏， 这一过程受一氧化氮抗炎作用的调节。在……里面 此外，人类SP-B选择性剪接的发育模式 肺及其剪接变异体与SP-B水平和新生儿肺的关系 疾病将会被确定。拟议的研究将利用这两种组织 文化和临床核心，并与项目6、4和 7.新的信息将使人们进一步了解 疏水性表面活性蛋白与肺发育和新生儿肺的关系 疾病。

(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.