SURFACTANT PROTEINS AND TYPE II CELL DIFFERENTIATION

遗传性肺表面活性物质缺乏症的研究进展 蛋白质B（SP-B），导致呼吸衰竭和严重形式的 婴儿支气管肺发育不良（BPD），表明这一关键作用 II型细胞分化中的蛋白质。 从SP-B分离的表面活性剂 缺陷型患者具有高表面张力和II型细胞结构， 功能异常，板层体发生失败， SP-C的加工 我们观察到胎儿肺上皮细胞 表达SP-B mRNA，但除非与糖皮质激素一起培养， 成熟蛋白和少量板层体。 根据这些观察，我们 假设成熟（8 kDa）SP-B蛋白的表达是一个关键的 成熟II型细胞分化中的事件，促进 多泡体形成板层体，其中SP-C的加工 发生。 本提案的目的是调查监管事件 在前体SP-B和SP-C的加工中的作用以及SP-B蛋白在 表面活性物质代谢具体目标1将评估发展 SP-B蛋白在表面活性剂中的合成和加工模式 新陈代谢. 具体目标1将评估发展模式， SP-B和SP-C蛋白的合成和加工以及 使用胎鼠和人肺外植体的板层体。 蛋白质 研究将使用具有表位特异性抗体的蛋白质免疫印迹， 以及脉冲追踪免疫沉淀方案，板层体将 使用共聚焦显微镜用膜标记抗体进行研究 免疫荧光和免疫电子显微镜。 目标2的研究将 检查对改变SP-B基因表达水平的反应。 SP-B mRNA 使用重组腺病毒， 在CMV启动子和激素控制下的正义或反义SP-B cDNA 将用于加速上皮细胞分化。 的目标 目的3是研究表面活性蛋白基因表达和磷脂 在患有肺病的婴儿的肺组织中的代谢。 SP-B缺乏， BPD和对照组织将作为外植体进行培养，用于分析 表面活性蛋白基因转录率和mRNA含量，蛋白质 加工和含量，以及表面活性剂磷脂的合成。 表面活性剂蛋白质也将在肺灌洗样品中进行测定， 早产儿，以确定是否SP-B发育缺陷 有助于呼吸窘迫和BPD的发生。 目标4将 确定重组SP-B表达在肺细胞中的作用， 患有SP-B遗传性缺陷的婴儿。 将开展研究， 在肺外植体中以及在免疫接种中作为异种移植物维持的组织中， 缺陷小鼠 该项目的研究将定义表达和 在发育过程中处理表面活性剂蛋白质， 了解SP-B在II型细胞分化中的作用， 功能 其结果将直接关系到基因的未来发展 SP-B遗传性缺乏性肺病的治疗 或其他原因。

The recent description of inherited deficiency of pulmonary surfactant protein B (SP-B), which results in respiratory failure and a severe form of bronchopulmonary dysplasia (BPD) in infants, suggests a key role of this protein in differentiation of type II cells. Surfactant isolated from SP-B deficient patients has high surface tension and type II cell structure and function is abnormal with failure of lamellar body genesis and incomplete processing of SP-C. We have observed that fetal lung epithelial cells express SP-B mRNA, but unless cultured with glucocorticoid, contain little mature protein and few lamellar bodies. Based on these observations, we hypothesize the expression of mature (8 kDa) SP-B protein is a critical event in differentiation of mature type II cells, promoting fusion of multivesicular bodies to form lamellar bodies where processing of SP-C occurs. The objective of this proposal is to investigate regulatory events in processing of precursor SP-B and SP-C and the role of SP-B protein in surfactant metabolism. Specific Aim 1 will evaluate the developmental pattern for synthesis and processing of SP-B protein in surfactant metabolism. Specific Aim 1 will evaluate the developmental pattern for synthesis and processing of SP-B and SP-C proteins and formation of lamellar bodies using fetal rat and human lung explants. The protein studies will use western immunoblotting with epitope specific antibodies as well as pulse-chase immunoprecipitation protocols, and lamellar bodies will be studied with membrane marker antibodies using confocal immunofluorescence and immuno electron microscopy. Studies of Aim 2 will examine responses to modifying levels of SP-B gene expression. SP-B mRNA content will be increased and decreased using recombinant adenoviruses with sense or antisense SP-B cDNA under control of the CMV promoter and hormones will be used to accelerate epithelial cell differentiation. The goal of aim 3 is to study surfactant protein gene expression and phospholipid metabolism in lung tissue from infants with lung disease. SP-B deficient, BPD and control tissue will be cultured as explants for analysis of surfactant protein gene transcription rate and mRNA content, protein processing and content, and synthesis of surfactant phospholipids. Surfactant proteins will also be assayed in lung lavage samples from premature infants to determine whether a developmental deficiency of SP-B contributes to occurrence of respiratory distress and BPD. Aim 4 will determine the effects of recombinant SP-B expression in lung cells from infants with an inherited deficiency of SP-B. Studies will be carried out in lung explants and with tissue maintained as xenografts in immune deficient mice. The studies of this project will define expression and processing of surfactant proteins during development and enhance our understanding of the role of SP-B in type II cell differentiation and function. Results will relate directly to future development of gene therapy for infants with lung disease due to inherited deficiency of SP-B or other causes.