REGULATION OF NORMAL HUMAN AIRWAY EPITHELIAL CULTURES

随着NHLBI授予1R01-HL33142，我们已经连续建立了 无血清培养上皮细胞传代培养，来自 人体气管。细胞对营养作用保持敏感。 维生素A(视黄醇)可促进分泌细胞分化。 另一方面，高浓度的钙(1.0-2.0 mm)， 似乎抑制分泌细胞分化并刺激细胞 低浓度(0.05-0.1 mM)有利于增殖 分泌细胞分化，增殖率低。这个 续期申请的主要目标是(1)进一步 分泌粘液糖蛋白的特性；(2)制备 分泌的单抗和多克隆抗体 糖蛋白；(3)鉴定呼吸道上皮细胞类型 体内合成这些糖蛋白；(4)鉴定唯一 维生素A和钙处理的培养物中的mRNA序列 文库消减杂交；(5)测定 钙对分化的快速影响是否涉及(A) 对质膜的影响和/或钙转运的变化， (B)蛋白质磷酸化的改变，(6)确定细胞是否 文化对文化的直接影响 神经药理药剂，以及(7)连续传播和 患者呼吸道上皮细胞培养的特征 患有囊性纤维症。 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的研究 培养对于确定粘液中的不规则性是必不可少的 分泌物与许多疾病有关，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.