NEUROPEPTIDES AND LUNG DEVELOPMENT

早产儿肺不成熟的发病率和死亡率 这是一个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，特别是蛙皮素样多肽(BLP)，在调节正常 胎儿肺发育基本上还没有被研究过。蓝斑狼疮很可能 参与正常的肺部发育。我们之前演示过 BLP mRNAs在妊娠中期人胎肺中的瞬时表达 与航空公司的增长同步。宫内BLP给药 加速小鼠胎肺生长成熟与内源性肺 用抗BLP单抗(MAb)阻断成熟。这个 细胞表面酶CD10/中性内肽酶24.11(CD10/NEP)已被 发现可以水解和灭活BLP，BLP是 正常的支气管上皮细胞、肺成纤维细胞和许多小的 肺癌(SCLC)。对CD10/NEP的特异性抑制作用 SCH32615促进人胎肺器官培养中胎肺的生长 在宫内发育的小鼠胎肺中生长和成熟。所有的 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。拟议的研究将集中于 参与肺调节的细胞和分子事件 BLPS和CD10/NEP的形态发生、生长和成熟我们计划 分析BLPS和/或CD10/NEP抑制对肺发育的影响 第一次是在子宫中，第二次是在肺器官培养过程中 早期器官发生(分枝、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的反应，以及潜在的协同作用 将探索不同的药理制剂之间的关系。第三，我们将 对观察到的基因进行定位和定量表达 对小鼠体内肺发育的影响。BLP、BLP受体和 CD10/NEP转录本和蛋白质将通过原位定位 杂交和免疫过氧化物酶分析，然后与BLP相关联 受体放射自显影和NEP酶组织化学。谈话稿 水平将使用RNAase保护和/或半 定量RT-PCR。第四，BLP效果的蜂窝要求将 在分离和重组的胎肺细胞培养中进行评估。如果 需要像成纤维细胞这样的中间细胞，我们将 确定是否需要细胞接触或扩散因素 可以起到调节作用。第五，我们将确定同源异型盒基因是否 参与BLP对分枝和细胞分化的影响 RNA分析和反义策略。最后，我们将研究胎肺 原发PNEC增生转基因小鼠的发育。如果有的话 在这些小鼠身上观察到的效应可以用BLP反义寡核苷酸来阻断 策略，这将确认BLPS是主要的PNEC衍生肽(S) 参与肺部发育。了解其作用机制 神经肽和CD10/NEP对旁分泌/自分泌的调节作用 促进这些药物在临床上的广泛应用 医药。

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.