THE ROLE OF APOPTOSIS IN CF PSEUDOMONAL LUNG INFECTION

遗传性疾病囊性纤维化的临床特征是慢性肺部感染，特别是铜绿假单胞菌感染。拟议项目的总体目标是进一步深入了解囊性纤维化跨膜传导调节因子(CFTR)基因产物的缺陷与CF患者铜绿假单胞菌肺部感染的发病机制之间的联系。在CF细胞培养模型中使用TUNEL法进行的初步研究表明，铜绿假单胞菌可以诱导正常肺细胞的凋亡，但不能诱导CF细胞的凋亡。本项目的具体目标是阐明细胞凋亡在铜绿假单胞菌与呼吸道上皮细胞相互作用中的作用。其具体目的有两个：1)利用TUNEL染色、流式细胞仪分析和正常和CF肺细胞的共聚焦显微镜来表征感染铜绿假单胞菌后对细胞培养和小鼠CF模型的凋亡诱导，A.具体地说，详细描述感染诱导凋亡的时间过程，B.建立铜绿假单胞菌内化在诱导细胞凋亡中的作用，以及C.确定caspase在感染诱导的细胞凋亡中的作用，以及2)使用DNA微阵列技术比较感染铜绿假单胞菌前后表达不同CFTR等位基因的肺细胞中调节细胞凋亡的基因的表达。确定野生型和deltaF508CFTR基因在感染铜绿假单胞菌后诱导肺细胞凋亡中的表达水平，以及B.确定感染诱导基因表达的时程。这种方法将允许同时监测数百个基因的表达水平，潜在地识别和暗示以前没有被认为是呼吸系统细胞-假单胞菌相互作用过程中参与者的细胞通路。因此，我们建议研究细胞凋亡在假瘤肺部感染发病机制中的作用，最终目的可能是根据这些研究的发现制定新的治疗策略。这个项目的完成有望为申请者提供必要的经验，使其成为假瘤病原学领域的独立研究者，并对CF肺部疾病的发病机制提供进一步的见解。

The clinical hallmark of the genetic disorder cystic fibrosis (CF) is chronic pulmonary infection, particularly with Pseudomonas aeruginosa. The global objective of the proposed project is to gain further insight into the link between defects in the CF gene product, the cystic fibrosis transmembrane conductance regulator (CFTR), and the pathogenesis of pulmonary infections with P. aeruginosa in CF patients. Preliminary studies in a CF cell culture model using the TUNEL assay indicates that P. aeruginosa induces apoptosis in normal, but not CF, lung cells. The specific objective of this project is to delineate the role of apoptosis in the interaction of P. aeruginosa with respiratory epithelium. The specific aims are twofold: 1) characterize the induction of apoptosis in cell culture and mouse models of CF following infection with P. aeruginosa, using TUNEL staining, FACS analysis and confocal microscopy of normal and CF lung cells, a. specifically, to detail the time-course of infection induced apoptosis, b. establish the role of internalization of P. aeruginosa in the induction of apoptosis, and c. determine the role of caspases in the infection-induced apoptosis, and 2) compare the expression of genes regulating apoptosis in lung cells expressing differing Cftr alleles, both before and after infection with P. aeruginosa, using DNA-microarray technology, a. specifically, to identify the expression levels of genes instrumental in orchestrating apoptosis in both wild-type and deltaF508 CFTR expressing lung cells after infection with P. aeruginosa, and b. determine the time course of expression of infection induced genes. This approach will allow simultaneous monitoring of the expression levels of hundreds of genes, potentially identifying and implicating cellular pathways previously unappreciated as participants in the process of respiratory cell-pseudomonal interaction. Thus, we propose to investigate the role of apoptosis in the pathogenesis of pseudomonal pulmonary infection in CF with the ultimate goal of perhaps formulating new treatment strategies based on the findings of these studies. Completion of this project promises to provide the experience necessary for the applicant to initiate a career as an independent investigator in the field of pseudomonal pathogensis, and to provide further insight in the pathogenesis of CF lung diseases.