REGULATION OF PULMONARY ELASTIN PRODUCTION BY RETINOIDS

破坏性炎症性肺病 肺气肿和支气管扩张会不可逆转地改变弹性 肺的结构蛋白弹性蛋白的降解所产生的特性。 因为几乎所有正常肺中的弹性蛋白都是在 早期，研究调节弹性蛋白合成的因素和 沉积，并最终修复弹性蛋白网络 在疾病中受损的。假设：围产期肺含有一种补给 维甲酸，可在最大牙槽期使用 隔弹性蛋白合成。维甲酸，尤其是维甲酸 (RA)可促进间质肺成纤维细胞合成弹性蛋白 (Lif)并调节所需的弹性蛋白合成的增加 用于正常的肺泡间隔形成。初步研究表明， 视黄酸和维甲酸受体-γm RNA及 新生大鼠肺成纤维细胞蛋白质的时态变化 这表明它们可以通过以下方式帮助启动弹性蛋白合成的增加 这些细胞。更多研究表明，类风湿关节炎可增加弹性蛋白 由培养的新生儿LIF产生，并在 抄写。拟议研究的主要目标是检查 类风湿性关节炎影响弹性蛋白合成的分子机制 在正常的肺泡发育期间。习得与新陈代谢 将检查肺组织和分离的LIF中维甲酸的含量，以评估 内源性肺贮物的利用。基础和RA- 诱导多种维甲酸受体(RAR)和 维甲酸X受体(RXR)基因将在培养的大鼠LIF中进行研究 从RAR-γ基因缺失小鼠分离的LIF中。RAR和RXR 将使用核糖核酸酶保护对mRNA和蛋白质进行定量 检测方法和免疫印迹方法。一家独大的影响 弹性蛋白表达的RAR阴性突变将在 培养的细胞。弹性蛋白mRNA，不溶性弹性蛋白积聚，以及 将研究携带基因缺失的小鼠的肺泡生长 RAR-伽马和/或RXR-α。这些效应的分子细节 RA对弹性蛋白基因的影响将通过缺失分析和 两个潜在类风湿反应元件(稀有)的致突变作用 大鼠弹性蛋白基因的5‘侧翼区域。电泳率 移位分析将被用来证明RA反应的增加 在培养的LIF和In中，核蛋白与这些元素的结合 发育中的肺。一种连接介导的聚合酶链式反应 将被用来评估蛋白质与这些假定的稀有蛋白的结合 在肺发育过程中体内的弹性蛋白基因，以及对 外源性RA。阐明弹性蛋白合成的机制 在肺泡中启动将提供新的信息 适用于特发性肺纤维化和支气管肺 发育不良。

Destructive inflammatory lung diseases lung diseases such as emphysema and bronchiectasis can irreversibly alter the elastic properties of the lung by degradation of the structural protein elastin. Since virtually all of the elastin in the normal lung is produced during early like, studying the factors which regulate elastin synthesis and deposition, and to ultimately repair the elastin network that is damaged in disease. Hypothesis: The perinatal lung contains a supply of retinoids, that it may use during the period of maximal alveolar septal elastin synthesis. Retinoids, and in particular retinoic acid (RA), may promote elastin synthesis by interstitial lung fibroblasts (LIF) and modulate the increase in elastin synthesis that is required for normal alveolar septal formation. Preliminary studies show that the quantities of RA and retinoic acid receptor-gamma mRNA and protein in neonatal rat lung fibroblasts change in a temporal pattern that suggests they could help initiate an increase in elastin synthesis by these cells. Additional studies show that RA increases elastin production by cultured neonatal LIF and acts at the level of transcription. The major goal of the proposed research is to examine the molecular mechanisms by which RA may influence elastin synthesis during normal alveolar development. The acquisition and metabolism of retinoids by lung tissue and isolated LIF will be examined to assess the utilization of endogenous pulmonary stores. The basal and RA- induced expression of the various retinoic acid receptor (RAR) and retinoid-X receptor (RXR) genes will be studied in cultured rat LIF and in LIF isolated from RAR-gamma null mice. RAR and RXR mRNA and protein will be quantitated using ribonuclease protection assays and immunoblotting, respectively. The effects of a dominant negative RAR mutation of elastin expression will be examined in cultured cells. Elastin mRNA, insoluble elastin accumulation, and alveolar growth will be studied in mice bearing gene deletions for RAR-gamma and/or RXR-alpha. The molecular details of the effects of RA on the elastin gene will be elucidated by deletional analysis and mutagenesis of two potential RA response elements (RARE) within the 5' flanking region of the rat elastin gene. Electrophorectic mobility shift assays will be used to demonstrate RA-responsive increases in the binding of nuclear proteins to these elements, in cultured LIF and in the developing lung. A ligation-mediated polymerase chain reaction will be used to evaluate binding of proteins to these putative RARE in the elastin gene in vivo during lung development, and in response to exogenous RA. Elucidation of mechanisms whereby elastin synthesis is initiated in the alveoli would provide novel information that may also be applicable to idiopathic pulmonary fibrosis and bronchopulmonary dysplasia.