IDENTIFICATION OF BIOCHEMICAL ABNORMALITIES IN PCD CILIA

原发性纤毛运动障碍（PCD）是一种以呼吸道纤毛超微结构异常为特征的遗传性疾病。呼吸纤毛的缺陷被认为会导致纤毛黏液清除受损，受影响的个体会反复出现呼吸道感染，包括鼻炎、鼻窦炎、支气管炎和肺炎。此外，许多PCD患者还患有慢性中耳炎，男性往往不育。目前，对于PCD没有有效的治疗方法，而且这种疾病的遗传基础是未知的。这项建议的长期目标是确定PCD的遗传基础和了解疾病的发病机制。为了实现这些目标，提出了以下具体目标：1：鉴定从培养的PCD细胞分离的纤毛中缺失或改变的蛋白质。2：开发和表征PCD细胞纤毛中改变蛋白的分子和生化探针。目的：确定PCD患者纤毛中缺失或改变蛋白的基因编码是否发生突变。从正常个体和PCD个体分离的气道上皮细胞将在体外培养，使用允许细胞分化成良好纤毛上皮的技术。细胞蛋白将通过结合标记的前体进行放射性标记，纤毛将被分离。纤毛蛋白将使用一维和二维聚丙烯酰胺凝胶电泳进行比较。PCD细胞纤毛中改变或缺失的蛋白质将被分离并通过质谱法鉴定。抗体和cDNA探针将针对鉴定的蛋白质。这些探针将用于确定蛋白质的基因编码是否发生突变，或者PCD纤毛中的蛋白质是否由于另一个基因的突变而发生改变。这些研究将在单个蛋白质水平上确定PCD患者纤毛的缺陷。最终，这些研究将确定导致某些PCD病例的突变，并将增加我们对突变如何导致疾病的理解。这一信息可能会改善这种疾病的诊断和治疗，包括基因治疗的可能性。进一步研究这些蛋白在纤毛的组装和功能中的作用，也可能导致改善其他气道疾病的治疗。

Primary ciliary dyskinesia (PCD) is an inherited disease which is characterized by various ultrastructural abnormalities in the cilia lining the respiratory tract. The defects in respiratory cilia are believed to result in impaired mucociliary clearance, and affected individuals suffer from recurrent respiratory infections, including rhinitis, sinusitis, bronchitis and pneumonia. In addition, many PCD patients also suffer from chronic otitis media, and males are frequently infertile. Currently, there is no curative treatment available for PCD, and the genetic basis of the disease is unknown. The long-term objectives of this proposal are to identify the genetic basis of PCD and to understand the pathogenesis of the disease. To achieve these goals, the following specific aims are proposed: 1: To identify protein(s) which are absent or altered in cilia isolated from cultured PCD cells. 2: To develop and characterize molecular and biochemical probes specific for the protein(s) which are altered in the cilia of PCD cells. 3: To determine if the gene coding for the protein(s) absent or altered in cilia from a PCD patient is mutated. Airway epithelial cells isolated from normal individuals and individuals with PCD will be cultured in vitro using techniques which allow the cells to differentiate into a well-ciliated epithelium. Cellular proteins will be radioactively labelled by the incorporation of labelled precursors and cilia will be isolated. The ciliary proteins will be compared using one- and two-dimensional polyacrylamide gel electrophoresis. Proteins which are altered or absent in the cilia from the PCD cells will be isolated and identified by mass-spectrometry. Antibodies and cDNA probes will be developed against the identified proteins. These probes will be used to determine if the gene coding for the protein is mutated, or if the protein appears altered in PCD cilia due to a mutation in another gene. These studies will identify defects in the cilia of PCD patients at the level of individual proteins. Ultimately, these studies will identify the mutation responsible for some cases of PCD, and will increase our understanding of how the mutation leads to disease. This information may lead to improvements in the diagnosis and therapy of this disease, including the possibility of gene therapy. Further studies of the role of these proteins in the assembly and function of cilia may also result in improved treatment for other air way diseases.