NEUROPEPTIDES AND LUNG DEVELOPMENT

神经肽与肺部发育

• 批准号: 2226133
• 负责人: Mary E. Sunday
• 金额: $ 28.28万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2226133
• 关键词: RNase protection assay; bombesin; cell differentiation; embryo /fetus; embryo /fetus cell /tissue; endopeptidases; gene expression; genetically modified animals; glucocorticoids; growth /development; growth factor; histogenesis; homeobox genes; hormone regulation /control mechanism; hyperplasia; immunoperoxidase; in situ hybridization; laboratory mouse; laboratory rabbit; lung; mammalian embryology; neuropeptides; organ culture; polymerase chain reaction; protease inhibitor

Morbidity and mortality due to lung immaturity in premature infants is a continuing major health problem. The role of endogenous neuropeptides derived from pulmonary neuroendocrine cells (PNECs) and pulmonary nerve fibers, in particular bombesin-like peptides (BLPs), in modulating normal fetal lung development has been essentially unexplored. BLPs are likely to participate in normal lung development. We previously demonstrated transient expression of BLP mRNAs in mid-gestation human fetal lung in parallel with growth of the airways. In utero BLP administration accelerated murine fetal lung growth and maturation and endogenous lung maturation was blocked by an anti-BLP monoclonal antibody (mAb). The cell surface enzyme CD10/neutral endopeptidase 24.11 (CD10/NEP) has been found to hydrolyze and inactivate BLPs, which are important mitogens for normal bronchial epithelial cells, pulmonary fibroblasts, and many small cell carcinomas of the lung (SCLCs). Specific inhibition of CD10/NEP by SCH32615 potentiated fetal lung growth in human fetal lung organ cultures and both growth and maturation in murine fetal lung in utero. All of these paracrine effects on fetal lung organ growth and maturation were blocked by bombesin receptor antagonists. The proposed study will focus on cellular and molecular events involved in the regulation of lung morphogenesis, growth and maturation by BLPs and by CD10/NEP. We plan to analyze effects of BLPs and/or CD10/NEP inhibition on lung development in fetal mice first in utero and second in lung organ cultures during early organogenesis (branching morphogenesis and growth) and later fetal lung growth and maturation. These effects will be compared with responded to glucocorticoids and growth factors, and potential synergism between diverse pharmacological agents will be explored. Third, we will localize and quantitate expression of genes regulating the observed effects on mouse lung development in vivo. BLP, BLP receptor and CD10/NEP transcripts and proteins will be localized using in situ hybridization and immunoperoxidase analyses, then correlated with BLP receptor autoradiography and NEP enzyme histochemistry. Transcript levels will be quantitated using RNAase protection and/or semi- quantitative RT-PCR. Fourth, cellular requirements for BLP effects will be assessed in isolated and reconstituted fetal lung cell cultures. If an intermediary cell such as the fibroblast is required, we will determine whether cell contact is required or whether a diffusible factor can mediate the effect. Fifth, we will determine whether homeobox genes are involved in BLP effects on branching and cell differentiation using RNA analyses and antisense strategies. Finally, we will study fetal lung development in transgenic mice with primary PNEC hyperplasia. If any effects observed in these mice can be blocked using BLP antisense strategies, this would confirm BLPs as the major PNEC-derived peptide(s) involved in lung development. Understanding mechanisms of paracrine/autocrine regulation by neuropeptides and CD10/NEP would facilitate a comprehensive application of these agents in clinical medicine.

早产婴儿的肺部不成熟引起的发病率和死亡率是 持续的重大健康问题。 内源性神经肽的作用 源自肺神经内分泌细胞（PNEC）和肺神经 调节正常 胎儿肺发育基本上没有探索。 BLP可能是 参加正常的肺发育。 我们以前证明了 BLP mRNA在妊娠中期人胎儿肺中的瞬时表达 与气道的生长平行。 在子宫内BLP管理 加速鼠胎儿肺的生长和成熟和内源性肺 通过抗BLP单克隆抗体（MAB）阻止成熟。 这 细胞表面酶CD10/中性内肽酶24.11（CD10/NEP） 发现可以水解和灭活BLP，这是重要的有丝分裂剂 正常支气管上皮细胞，肺成纤维细胞和许多小 肺的细胞癌（SCLCS）。 特定抑制CD10/NEP SCH32615人类胎儿肺器官培养中增强的胎儿肺生长 以及子宫内鼠胎儿肺的生长和成熟。 所有人 这些对胎儿肺器官生长和成熟的旁分泌作用是 被孟买受体拮抗剂阻塞。拟议的研究将重点 在与肺调节有关的细胞和分子事件上 BLP和CD10/NEP的形态发生，生长和成熟。 我们计划 分析BLP和/或CD10/NEP抑制对肺发育的影响 在子宫内首先在胎儿小鼠中，在肺部培养物中第二 早期器官发生（分支形态发生和生长）和后来的胎儿 肺部生长和成熟。 这些效果将与 对糖皮质激素和生长因子以及潜在的协同作用做出了反应 将探索不同的药理学剂之间。 第三，我们会的 定位和定量调节观察到的基因的表达 对体内小鼠肺发育的影响。 BLP，BLP受体和 CD10/NEP成绩单和蛋白质将使用原位定位 杂交和免疫过氧化物酶分析，然后与BLP相关 受体放射自显影和NEP酶组织化学。 成绩单 将使用RNAase保护和/或半定量水平 定量RT-PCR。 第四，BLP效应的细胞要求将 在分离和重构的胎儿肺细胞培养物中进行评估。 如果 需要一个中介细胞，例如成纤维细胞，我们将 确定是否需要细胞接触或是否可以扩散因子 可以调节效果。 第五，我们将确定同型基因基因是否 使用BLP对分支和细胞分化的影响 RNA分析和反义策略。 最后，我们将研究胎儿肺 主要PNEC增生的转基因小鼠的发育。 如果有的话 在这些小鼠中观察到的效果可以使用BLP反义阻止 策略，这将确认BLP是主要PNEC衍生的肽 参与肺发育。 了解的机制 神经肽和CD10/NEP的旁分泌/自分泌调节将 促进这些药物在临床上的全面应用 药品。