THE ROLE OF APOPTOSIS IN CF PSEUDOMONAL LUNG INFECTION

遗传性疾病囊性纤维化（CF）的临床标志是慢性肺部感染，特别是铜绿假单胞菌。该项目的全球目标是进一步了解CF基因产物、囊性纤维化跨膜传导调节因子（CFTR）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试验的初步研究表明，铜绿假单胞菌诱导正常肺细胞凋亡，而不是CF肺细胞。本项目的具体目的是描述细胞凋亡在铜绿假单胞菌与呼吸道上皮相互作用中的作用。具体目标有两个：1)利用TUNEL染色、FACS分析和共聚焦显微镜对正常肺细胞和CF肺细胞感染后的细胞培养和小鼠模型进行诱导凋亡的表征，a.详细描述感染诱导凋亡的时间过程，b.确定P. aeruginosa内化在诱导细胞凋亡中的作用，c.确定caspase在感染诱导细胞凋亡中的作用。2)利用dna芯片技术，比较P. aeruginosa感染前后表达不同Cftr等位基因的肺细胞中调控凋亡基因的表达情况。a.具体而言，确定P. aeruginosa感染后野生型和deltaF508 Cftr肺细胞中调控凋亡基因的表达水平；b.确定感染诱导基因表达的时间过程。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未被发现的呼吸细胞-假单胞菌相互作用过程参与者的细胞途径。因此，我们建议研究细胞凋亡在CF假单胞菌肺部感染发病机制中的作用，最终目标可能是根据这些研究结果制定新的治疗策略。本项目的完成有望为申请人在假单胞菌病原学领域开启独立研究者的职业生涯提供必要的经验，并为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.