Genomic/proteomic analysis of human salivary glands

子项目3的总体目标是比较正常人腮腺和特发性口干患者腮腺组织中蛋白质和转录本的表达。总的假设是，特发性口干是由关键调节蛋白或效应蛋白表达的变化引起的。为了鉴定人腮腺特异性蛋白，在Specific Aim 1中，我们将创建一个人腮腺的表达序列标签（EST）数据库。为了实现这一目标，我们将从正常成人腮腺中构建一个定向cDNA文库。为了实现这一目标，我们将从正常成人腮腺组织中构建一个定向cDNA文库，并随机选择克隆的5'端进行测序。然后将序列与可用的数据库进行比较。我们假设特发性口干是由于关键调节蛋白或效应蛋白表达的变化导致体液分泌失调。为了验证这一假设，在具体目标2中：我们将使用微阵列和PCR扩增转录本的直接测序，比较正常腺体和特发性口干个体的转录本表达。作为对特异性目标1中概述的EST数据库的补充，我们在特异性目标3中建议绘制正常人腮腺和下颌下腺/舌下腺分泌的蛋白质谱。二维凝胶电泳将用于分离蛋白质，免疫印迹、Edman降解和基质辅助激光解吸/电离飞行时间（MALDI/TOF）质谱将用于鉴定蛋白质。我们假设，这种“正常”特征与从特发性口干患者身上收集的唾液所获得的特征之间存在差异，而特发性口干患者的唾液流速看似“正常”。在表达水平上表现出差异的蛋白质将成为维持口腔润滑和水合状态的功能角色的候选者。综上所述，这些方法将使我们能够识别导致口腔干燥的关键调节或效应蛋白的潜在缺陷。

The overall goal of Subproject 3 is to compare the expression of proteins and transcripts in normal human parotid gland with parotid tissue derived from individuals with idiopathic dry mouth. The overall hypothesis is that idiopathic dry mouth is caused by a change in the expression of key regulatory or effector proteins. To identify human parotid gland-specific proteins, in Specific Aim 1: we will create an expressed-sequence-tag [EST] data base of the human parotid gland. To accomplish this goal, we will construct a directional cDNA library from normal human adult parotid gland. To accomplish this goal, we will construct a directional cDNA library from normal human adult parotid gland tissue and will sequence the 5' end of randomly selected clones. Sequences will then be compared against available databases. We hypothesize that idiopathic dry mouth results from a change in the expression of key regulatory or effector proteins leading to dysregulation of fluid production. To test this hypothesis, in Specific Aim 2: we will compare the expression of transcripts in normal glands with those derived from individuals with idiopathic dry mouth using microarrays and direct sequencing of PCR amplified transcripts. As a complement to the EST database outlined in Specific Aim 1, we propose in Specific Aim 3, to map the spectrum of proteins which are secreted by the normal human parotid and submandibular/sublingual glands. Two dimensional gel electrophoresis will be used to separate proteins and combinations of immunoblotting, Edman degradation and matrix-assisted laser desorption/ionization time-of-flight (MALDI/TOF) mass spectrometry will be used to identify the proteins. We hypothesize that differences will be observed between this "normal" profile and one obtained using saliva collected from subjects with idiopathic dry mouth, who present with seemingly "normal" flow rates. Proteins displaying variance in their level for expression will be candidates for functional roles involved in maintaining the oral cavity in a lubricated and hydrated state. Taken together, these approaches will allow us to identify underlying defects in key regulatory or effector proteins which lead to oral dryness.