NEUROPEPTIDES AND LUNG DEVELOPMENT

早产儿肺不成熟的发病率和死亡率 持续的重大健康问题。 内源性神经肽的作用 来源于肺神经内分泌细胞（PNEC）和肺神经 纤维，特别是蛙皮素样肽（BLP），在调节正常 胎儿肺的发育基本上还未被探索。 BLP很可能 参与肺的正常发育 我们先前表明 BLP基因在人胎肺中的瞬时表达 与气道的生长平行。 子宫内BLP给药 促进小鼠胎肺生长和成熟以及内源性肺 成熟被抗BLP单克隆抗体（mAb）阻断。 的 细胞表面酶CD 10/中性内肽酶24.11（CD 10/NEP）已被 发现水解和水解BLP，这是重要的有丝分裂原， 正常支气管上皮细胞、肺成纤维细胞和许多小的 肺癌（SCLC）。 CD 10/NEP的特异性抑制 SCH 32615在人胎肺器官培养中促进胎肺生长 以及鼠胎肺在子宫内的生长和成熟。 所有 这些旁分泌对胎儿肺器官生长和成熟的影响是 被蛙皮素受体拮抗剂阻断。拟议的研究将侧重于 参与肺调节的细胞和分子事件 通过BLP和通过CD 10/NEP的形态发生、生长和成熟。 我们计划 分析BLP和/或CD 10/NEP抑制对肺发育的影响 在胎鼠中，首先在子宫内，其次在肺器官培养物中， 早期器官发生（分支形态发生和生长）和晚期胎儿 肺的生长和成熟。 这些影响将与 对糖皮质激素和生长因子有反应， 不同药物之间的关系将被探讨。 三是 定位和定量基因的表达， 对小鼠体内肺发育的影响。 BLP、BLP受体和 CD 10/NEP转录物和蛋白质将使用原位杂交定位。 杂交和免疫过氧化物酶分析，然后与BLP 受体放射自显影和NEP酶组织化学。 转录物 水平将使用RNA酶保护和/或半定量 定量RT-PCR。 第四，BLP效应的细胞要求将 在分离和重建的胎肺细胞培养物中进行评估。 如果 需要一个中间细胞，如成纤维细胞，我们将 确定是否需要电池接触或是否需要扩散因子 可以调节效果。 第五，我们将确定同源异型盒基因是否 参与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 在原发性PNEC增生的转基因小鼠中的发育。 如果有的 在这些小鼠中观察到的作用可以用BLP反义核酸阻断 这将证实BLP是主要的PNEC衍生肽 参与肺的发育。 了解的机制 神经肽和CD 10/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

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.