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。 拟议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发病机制之间的联系。 使用TUNEL测定法在CF细胞培养模型中的初步研究表明，铜绿假单胞菌诱导正常肺细胞而非CF肺细胞的凋亡。 本项目的具体目标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）使用正常和CF肺细胞的TUNEL染色、FACS分析和共聚焦显微镜来表征在用铜绿假单胞菌感染后细胞培养物和CF小鼠模型中细胞凋亡的诱导，a.具体地，详细描述感染诱导的细胞凋亡的时间过程，B.确立铜绿假单胞菌的内化在诱导细胞凋亡中的作用，以及c.确定半胱天冬酶在感染诱导的细胞凋亡中的作用，和2）使用DNA微阵列技术比较在铜绿假单胞菌感染之前和之后表达不同Cftr等位基因的肺细胞中调节细胞凋亡的基因的表达，a.具体地，鉴定在用铜绿假单胞菌感染后在表达野生型和deltaF508 CFTR的肺细胞中协调细胞凋亡的基因的表达水平，和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，可能识别和暗示以前未被认识到的呼吸细胞假单胞菌相互作用过程中的参与者的细胞途径。 因此，我们建议调查的作用，细胞凋亡的假单胞菌肺部感染的发病机制，CF的最终目标，也许制定新的治疗策略的基础上，这些研究的结果。 该项目的完成将为申请人提供必要的经验，使其能够在假单胞菌致病领域作为独立研究者开展职业生涯，并进一步了解CF肺部疾病的发病机制。

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.