SEROUS CELL MALFUNCTION AND CYSTIC FIBROSIS LUNG DISEASE

大多数囊性纤维化（CF）患者死于慢性肺部感染， 但CFTR突变如何损害粘膜防御尚不清楚。的 有待检验的假设是，CF肺病开始是因为CFTR 突变破坏了浆液细胞分泌，从而剥夺了CF气道的 由浆液细胞分泌的富含脂质的液体，其表达最高的 气道细胞中CFTR的水平。初步数据显示， 细胞需要CFTR来分泌液体。浆液细胞功能障碍 将通过测量抗生素水平直接测试假设， 分泌物。这个项目是由一个模型浆液细胞系 （Calu-3细胞），特别是通过改善人气道1度培养物 其保留“纯”浆液细胞表型。目的1检验假设， 浆液性细胞1级培养物和Calu-3细胞表达丰富的 抗微生物剂，包括防御素和凝集素。半定量RT- PCR将用于测量溶菌酶，乳铁蛋白， 分泌成分，血清白细胞蛋白酶抑制剂（SLPI），人 防御素分子hBD-1和hBD-2，以及凝集素SP-A。目标2将 检验人气道浆液细胞不分泌 CF中的抗生素ELISA将用于定量释放 来自Calu-3细胞的抗菌剂，来自浆液细胞1度培养物的抗菌剂， 对照和CF受试者，以及正常和CF受试者的鼻灌洗液。 目的3检验CF人气道浆液细胞不 分泌液体。将使用双面电容探针方法 测量1度浆液细胞单层的液体分泌， 对照组和CF个体。还将测量腺体分泌物， 新鲜切下的气管和支气管中的恒定口径毛细血管， 对照组和CF个体。目的4将检验浆液性 细胞需要CFTR来分泌以响应增加的[Ca/2+]/i，并且 它们分泌HCO/3和Cl-的可变混合物。这将由以下人员进行测试： 拍摄电路电流测量，同位素通量和膜片钳。的 本项目预期的结果是，Ca/2+依赖的Cl-分泌 在CF中，浆液细胞的液体将大大减少， 减少了从气道表面到达气道表面的抗生素量， 腺体

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.