REGULATION OF NORMAL HUMAN AIRWAY EPITHELIAL CULTURES

通过NHLBI拨款1R01-HL33142，我们连续建立了 在无血清培养基中传代上皮细胞培养物，来自 人体气管 细胞对营养效应保持敏感 维生素A（视黄醇），增强分泌细胞分化。 另一方面，高浓度的Ca ++（1.0 - 2.0mM）， 似乎抑制分泌细胞分化并刺激细胞分化， 增殖，而低浓度（0.05 - 0.1 mM）有利于 分泌细胞分化和较低的增殖速率。 的 续期申请的主要目的是（1）进一步 表征分泌的粘液糖蛋白;（2）制备 抗分泌的 糖蛋白;（3）识别气道上皮细胞的类型 体内合成这些糖蛋白;（4）鉴定独特的 维生素A和Ca ++处理的培养物中的mRNA序列， cDNA文库的消减杂交;（5）确定 Ca ++对分化的快速作用是否涉及（a） 对质膜的影响和/或Ca ++转运的改变， (b)蛋白质磷酸化的改变，（6）确定细胞是否 文化对直接影响的反应 神经药理学试剂，和（7）连续传播和 表征患者气道上皮细胞培养物 囊性纤维化 细胞、生化和分子调控的阐明 正常人气管上皮细胞粘液分泌 为了确定粘液中的不规则性， 分泌物参与许多疾病，包括囊性 纤维化哮喘和炎症

With the NHLBI grant 1R01-HL33142, we have established serially passaged epithelial cell cultures in serum-free medium, from the human trachea. The cells remain sensitive to the trophic effects of vitamin A (retinol) that enhances secretory cell differentiation. High concentrations of Ca++ (1.0-2.0mM), on the other hand, appear to inhibit secretory cell differentiation and stimulate cell proliferation while low concentrations (0.05-0.1 mM) favor secretory cell differentiation and lower rate of proliferation. The major objectives of the renewal application are (1) to further characterize the secreted mucous glycoproteins; (2) prepare monoclonal and polyclonal antibodies to the secreted glycoproteins; (3) identify the types of airway epithelial cells in vivo which synthesize these glycoproteins; (4) identify unique mRNA sequences in vitamin A and Ca++ treated cultures by subtractive hybridization of cDNA libraries; (5) determine whether the rapid effects of Ca++ on differentiation involve (a) effects on plasma membrane and/or alterations in Ca++ transport, (b) alteration in protein phosphorylation, (6) determine if the cell cultures are responsive to the direct effects of neuropharmacological agents, and (7) serially propagate and characterize epithelial cell cultures from the airway of patients with cystic fibrosis. The elucidation of cellular, biochemical and molecular regulations of mucus secretion in normal human tracheal epithelial cell cultures is essential in order to define irregularities in mucus secretion involved in many disease conditions including cystic fibrosis, asthma and inflammation.