SEROUS CELL MALFUNCTION AND CYSTIC FIBROSIS LUNG DISEASE

浆液细胞功能障碍和囊性纤维化肺病

• 批准号: 6110948
• 负责人: JEFFREY J WINE
• 金额: $ 18.09万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6110948
• 关键词: antiserum; cell line; chloride channels; cystic fibrosis; defensins; lactoferrin; lysozyme; mucosal immunity; polymerase chain reaction; protease inhibitor; pulmonary surfactants; respiratory enzyme; respiratory epithelium; secretion

Most people with cystic fibrosis (CF) die from chronic lung infections, but it is unknown how CFTR mutations compromise mucosal defenses. The hypothesis to be tested is that CF lung disease begins because CFTR mutations disrupt serous cell secretion, thus depriving CF airways of the antibiotic-rich fluid secreted by serous cells, which express the highest levels of CFTR among airway cells. Preliminary data indicate that serous cells require CFTR for fluid secretion. The serous cell malfunction hypothesis will be directly tested by measuring antibiotic levels in secretions. This project is made possible by a model serous cell line (Calu-3 cells) and especially by improve human airway 1 degree cultures that retain a "pure" serous cell phenotype. Aim 1 test the hypothesis that serous cell 1 degree cultures and Calu-3 cells express abundant antimicrobials, including defensins and collectins. Semi-quantitative RT- PCR will be used to measure expression of mRNA for lysozyme, lactoferrin, secretory component, serum leukocyte protease inhibitor (SLPI), the human defensin molecules hBD-1 and hBD-2, and the collectin SP-A. Aim 2 will test the hypothesis that human airway serous cells do not secrete antibiotics in CF. ELISA will be used to quantify the release of antimicrobials from Calu-3 cells, from serous cell 1 degree cultures from control and CF subjects, and in nasal lavages from normal and CF subjects. Aim 3 tests the hypothesis that CF human airway serous cells do not secrete fluid. A double-sided capacitance probe method will be used to measure fluid secretion across the 1 degree serous cell monolayers from controls and CF individuals. Gland secretions will also be measured with constant bore capillaries in freshly excised trachea and bronchi from controls and CF individuals. Aim 4 will test the hypothesis that serous cells require CFTR to secrete in response to increase [Ca/2+]/i, and that they secrete a variable mixture of HCO/3 and Cl-. This will be tested by shot circuit current measurements, isotope fluxes and patch-clamping. The results expected for this project are that Ca/2+ dependent secretion of Cl and fluid by serous cells will be greatly diminished in CF, resulting in reduced volumes of antibiotics reaching the airway surface from the glands.

大多数囊性纤维化患者死于慢性肺部感染， 但目前尚不清楚cftr突变是如何影响粘膜防御的。这个 有待检验的假说是，CF肺部疾病的开始是因为CFTR 突变扰乱浆液细胞分泌，从而剥夺了CF呼吸道的 浆液细胞分泌的富含抗生素的液体，表达最高 呼吸道细胞间CFTR值的变化。初步数据表明，血清 细胞需要CFTR才能分泌液体。浆液性细胞功能障碍 假说将通过测量抗生素水平来直接检验 分泌物。这个项目是由一种模型浆液性细胞系实现的 (CALU-3细胞)，尤其是通过改善人呼吸道1度培养 保留“纯”浆液性细胞表型。目标1检验假设 浆液细胞1度培养和CALU-3细胞表达丰富 抗菌剂，包括防御素和胶原素。半定量RT- 用聚合酶链式反应检测溶菌酶、乳铁蛋白、 分泌成分，血清白细胞蛋白酶抑制物(SLPI)，人类 防御素分子HBD-1和HBD-2，以及集合素SP-A。目标2将 检验人类呼吸道浆液细胞不分泌的假设 卡介苗中的抗生素。将使用ELISA法来定量释放 来自CALU-3细胞的抗菌素，来自浆液性细胞的1度培养 对照组和CF受试者，以及正常受试者和CF受试者的鼻腔冲洗液。 AIM 3测试了人呼吸道浆液细胞不存在的假设 分泌液体。将使用双面电容探头方法来 测量1度浆液细胞单层的液体分泌量 对照和CF个人。腺体分泌物也将用 新鲜切取的气管和支气管内的恒定口径毛细血管 对照和CF个人。目标4将检验这一假说 细胞需要CFTR分泌以响应[Ca/2+]/i的增加，并且 它们分泌一种可变的HCO/3和Cl-的混合物。这将通过以下方式进行测试 短路电流测量、同位素通量和膜片钳。这个 该项目预期的结果是依赖于钙/2+的氯的分泌 而浆液细胞的液体会在CF中大大减少，从而导致 减少从呼吸道表面进入呼吸道表面的抗生素体积 腺体。