SURFACTANT PROTEINS AND TYPE II CELL DIFFERENTIATION

（申请人的摘要）出生时缺乏肺表面活性物质是一种严重的肺损伤。 肺损伤和长期肺病的主要原因，如 支气管肺发育不良（BPD）。严重的呼吸窘迫 遗传性缺乏表面活性蛋白-B（SP-B），在两个小鼠幼崽 和足月出生的婴儿，表明疏水性SP在 II型细胞的分化。在没有SP-B的情况下， 正常板层体形成以及SP-C加工不完全。 最近，SP-C的孤立性缺乏症已被描述在婴儿， 间质性肺病呼吸窘迫也发生在新生儿期 缺乏成熟SP-C和SP-B减少的BWB小牛，以及啮齿动物 呼吸窘迫和获得性SP-B/-C缺乏与肺 继发于博来霉素或感染（卡氏肺孢子虫和内毒素）的损伤。基于 根据这些和其他研究结果，本项目建议，SP-B、SP-C的合成 和板层体是紧密相连的， SP-C影响表面活性剂的功能。这项建议的目的是 为了表征人SP-C的生物合成途径，确定 SP-B和SP-C在板层体发生中的作用，并研究SP-B和SP-C在板层体发生中的作用。 肺病。目的：研究成熟SP-C在II型糖尿病中的表达， 体内和体外细胞分化与SP-B产生的关系 和板层体，并且还定义了靶向结构域和切割事件， SP-C处理。这些研究将利用抗体使SP-C成熟， 最近开发的用于经尿道诱导的II型细胞的培养系统 体外分化。目的II将研究的作用和相互作用 SP-B和SP-C在板层体形成和表面活性剂运输中的作用 组分使用SP缺乏的细胞培养模型。这些研究将 研究这一假设，即成熟SP-B的表达是两种疾病所必需的。 片层体形成和SP-C中间体的最终加工。 实验将在培养的II型细胞模型和SP-B或 - C基因表达将使用腺病毒表达选择性抑制， 反义mRNA。每种SP的加工和细胞内运输将被 使用表位特异性抗体，脉冲/追踪标记， 重组蛋白此外，还介绍了交替处理的工艺和效果 将测定剪接的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中SP-B和SP-C的表达 吸入一氧化氮治疗后据推测， SP-B和/或SP-C缺乏发生在患有严重BPD的婴儿中， 该过程由一氧化氮的抗炎作用调节。在 此外，人类SP-B可变剪接的发育模式 剪接变异体与SP-B水平和新生儿肺的关系 疾病将被确定。拟定研究将使用组织 文化和临床核心，并涉及与项目6，4和 7.新的信息将使人们进一步了解 肺发育和新生肺中疏水表面活性蛋白 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.