GROWTH AND REPAIR OF AIRWAY EPITHELIUM IN ASTHMA

哮喘气道上皮的生长和修复

• 批准号: 2378814
• 负责人: STEVEN R WHITE
• 金额: $ 26.53万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-05 至 1999-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2378814
• 关键词: G protein; actins; afferent nerve; asthma; bombesin; calcitonin gene related peptide; cell adhesion; cell growth regulation; cell migration; extracellular matrix proteins; flow cytometry; gastrin releasing peptide; guinea pigs; human tissue; laboratory rat; mixed tissue /cell culture; neuroendocrine system; neuropeptide receptor; neuropeptides; protein kinase C; respiratory epithelium; spinal ganglion; substance K; substance P; transcription factor; western blottings

Studies are propose to examine airway epithelial cell growth, migration and attachment to extracellular matrices. Denudation of the epithelium may lead to worsened airflow obstruction by loss of epithelium-derived factors which modulate airway tone, ineffective mucous transport and perturbations in airway fluid transport. Repair of the epithelium may be an important factor in restoring airway homeostasis, while inflammatory cells may perpetuate epithelial damage. Preliminary data suggest that neuropeptides secreted by airway sensory nerves and neuroendocrine bodies may enhance epithelium repair. Two major goals are identified in this proposal: 1) Determine the modulation of airway epithelial cell growth by neuropeptides derived from airway sensory afferent nerves and neuroendocrine cells. We will test the hypothesis that neuropeptides (such as substance P, bombesin, gastrin releasing peptide, calcitonin gene-related peptide, and neurokinin A) stimulate proliferation of epithelial cells. The mechanisms by which neuropeptides stimulate proliferation, including the events transducing the signal, will be examined. Additional experiments will examine the differentiation of epithelial cells after neuropeptide stimulation, and the potential physiologic significance of neuropeptide stimulation. Epithelial cells will be collected from human bronchial surgical specimens and guinea pig tracheas and grown in culture. Specific epithelial cell sub-types will be isolated by immunomagnetic separation. Epithelial cell proliferation will be measured by incorporation of bromodeoxyuridine into DNA and by direct cell count. Expression of nuclear transcription factors will be determined by northern blot assay. Activation of mitogen-associated protein kinase will be tested using a specific renaturation assay. Initial activation of epithelial cells will be measured by cyclic AMP assay and by inhibition of G-protein related signal transduction. Involvement of protein kinase C will be tested using specific inhibitors. 2) Determine the mechanisms by which neuropeptides derived from airway sensory afferent nerves and neuroendocrine cells modulate airway epithelial cell migration and adhesion. We will test the hypotheses that neuropeptides increase migration of epithelial cells and augment epithelial cell adhesion to extracellular matrix proteins. Specific mechanisms will be identified by which neuropeptides elicit migration and stimulate expression of specific adhesion receptors in airway epithelial cells, and studies will determine the sequence of events in airway epithelial cell migration through extracellular matrices. Migration will be measured by blindwell assay through matrix-impregnated porous filters. Cell attachment to extracellular matrix proteins will be measured by specific microplate assay. Specific adhesion receptor antagonists and antibodies will be used to determine mechanisms of attachment. The role of focal adhesion kinase in transducing the neuropeptide stimulus for migration will be examined. These experiments will clarify mechanisms by which sensory nerves promote the repair of airway epithelium in the recovery from injury or inflammation. Data derived from these studies should provide insight into mechanisms by which airway epithelium may be restored in human asthma.

提出了研究以检查气道上皮细胞生长，迁移 并附着在细胞外矩阵上。上皮的剥落 导致由于上皮衍生因素的丧失而导致气流阻塞恶化 调节气道音调，无效的粘液传输和扰动 在气道流体运输中。维修上皮可能很重要 恢复气道稳态的因素，而炎症细胞可能 永久损害。初步数据表明神经肽 气道感觉神经和神经内分泌体的分泌可能会增强 上皮修复。在此提案中确定了两个主要目标：1） 确定神经肽对气道上皮细胞生长的调节 源自气道感觉传入神经和神经内分泌细胞。我们 将检验以下假设：神经肽（例如物质P， 孟买，胃蛋白释放肽，降钙素基因相关肽和 Neurokinin a）刺激上皮细胞的增殖。机制 神经肽刺激增殖，包括事件 将检查信号的传递。其他实验将 检查神经肽后上皮细胞的分化 刺激以及神经肽的潜在生理意义 刺激。上皮细胞将从人支气管中收集 手术标本和豚鼠气管，并在培养中生长。具体的 上皮细胞亚型将通过免疫磁分离分离。 上皮细胞增殖将通过合并来测量 溴脱氧尿苷进入DNA和直接细胞计数。核的表达 转录因子将通过北印迹测定法确定。 将使用A的激活促丝裂料相关蛋白激酶的激活将使用A 特定的肾脏化测定法。上皮细胞的初始激活将 可以通过循环AMP测定和抑制G蛋白相关测量 信号转导。蛋白激酶C的参与将使用 特定抑制剂。 2）确定神经肽的机制 源自气道感觉传入神经和神经内分泌细胞 调节气道上皮细胞迁移和粘附。我们将测试 假设神经肽会增加上皮细胞的迁移和 增强上皮细胞对细胞外基质蛋白的粘附。 将通过哪些神经肽引起的特定机制确定 迁移并刺激特定粘附受体的表达 气道上皮细胞，研究将确定事件的序列 在气道上皮细胞通过细胞外矩阵迁移。 迁移将通过盲孔测定通过矩阵浸渍来衡量 多孔过滤器。细胞附着细胞外基质蛋白将是 通过特定的微板测定法测量。特异性粘附受体 拮抗剂和抗体将用于确定 依恋。局灶性激酶在转导的作用 将检查用于迁移的神经肽刺激。这些实验 将阐明感觉神经促进修复的机制 气道上皮在受伤或炎症中恢复。数据 从这些研究中得出的应提供有关机制的洞察力 气道上皮可以在人类哮喘中恢复。