Role of CFTR in Toxicant-Sensitivity of Innate Immunity

描述(由申请者提供)：在这个项目中，将在斑马鱼中研究CFTR在先天性免疫中的作用，以及它被砷破坏的作用。有证据表明，三磷酸腺苷结合盒(ABC)转运蛋白，囊性纤维化跨膜电导调节因子(CFTR)与先天性免疫反应有关，CFTR突变导致人类对铜绿假单胞菌肺部感染的易感性增加。砷参与了天然免疫反应的紊乱，并被证明能抑制异地叉尾轮虫CFTR的表达，然而，这种环境毒物的作用与天然免疫中CFTR功能的破坏之间的直接联系尚未建立。为了检验这些假设，这项提议的具体目标是： 1.探讨重金属毒物砷暴露与机体天然免疫反应活性的关系 2.斑马鱼CFTR基因表达与先天免疫功能关系的研究 为了研究环境毒物在斑马鱼对假单胞菌感染的易感性中的作用，将利用病原体铜绿假单胞菌进行感染研究，并确定砷是否影响先天性免疫反应和改变对感染的抵抗力模式。将进行呼吸爆发测试，以检查砷处理鱼的先天免疫功能。斑马鱼cftr基因的表达将被吗啉反义寡核苷酸抑制，由此产生的cftr变异体将不再通过cftr结合铜绿假单胞菌IPS，随后将暴露于砷中并评估呼吸爆发活性。此外，还将从暴露在砷中的斑马鱼胚胎中提取RNA，并通过定量RT-PCR来确定处理对CFTR表达的影响。

DESCRIPTION (provided by applicant): In this project, the role of CFTR in innate immunity, and its disruption by arsenic, will be examined in the zebrafish. There is evidence that the ATP-binding cassette (ABC) transporter protein, cystic fibrosis transmembrane conductance regulator (CFTR), is linked to the innate immune response and that mutations in CFTR result in increased susceptibility to lung infection in humans by Pseudomonas aeruginosa. Arsenic has been implicated in the perturbation of innate immune response and has been shown to inhibit the expression of CFTR in the killifish, Fundulus heteroclitus, however, a direct link between the action of this environmental toxicant and the disruption of CFTR function in innate immunity has not been established. In order to test these hypotheses, the SPECIFIC AIMS of this proposal are: 1. To investigate the relationship between exposure to the heavy metal toxicant, arsenic, and the activity of the innate immune response 2. To determine the relationship between CFTR expression and innate immune function in the zebrafish To examine the role of environmental toxicants in susceptibility to Pseudomonas infection in the zebrafish, the pathogen P. aeruginosa will be used to carry out infection studies and to determine if arsenic affects the innate immune response and alters patterns of resistance to infection. Respiratory burst assays will be performed to examine innate immune function in arsenic-treated fish. Zebrafish CFTR gene expression will be knocked down by a morpholino antisense oligonucleotide, and the resulting CFTR morphants, which will no longer bind P. aeruginosa IPS via CFTR, will subsequently be exposed to arsenic and evaluated for respiratory burst activity. In addition, RNA will be extracted from zebrafish embryos exposed to arsenic and the effect of treatment on CFTR expression will be determined by quantitative RT-PCR.