Genes & Proteins of Urothelial-ECM Interaction

描述（由申请人提供）：尿道鞘内衬膀胱，通过保持不可渗透的屏障，保护膀胱免受尿液中的细菌和有毒或致癌物质的影响。虽然这功能非常好，但它在膀胱癌，间质性膀胱炎以及可能的其他疾病中失败。了解膀胱尿道的生长、分化及功能的发挥，可为膀胱疾病的诊断和治疗提供重要线索。虽然尿道上皮可以培养，但大多数模型未能充分模拟尿道上皮和下层基质之间的相互作用。两者共同构成了一个复杂的、相互作用的系统。该提案描述了满足PAR要求的研究，以开发细胞选择性工具，特别是项目（8）鉴定细胞特异性标志物，以帮助研究正常和恶性组织中的上皮-基质相互作用，其次是项目（5）发现指示个体细胞类型的健康或质量的生物标志物，以及项目（1）发现对个体细胞类型具有选择性的基因。用于在正常和癌症修饰的基质上生长作为三维人工组织的尿路上皮和作为人工肿瘤的膀胱癌的新方法为发现参与定义正常和恶性尿路上皮的基因和蛋白质提供了基础。在第一个发现阶段，微阵列和一种新的蛋白质组学方法与2-D色谱将结合创新的生物信息学方法，以确定候选基因和蛋白质。将建立一个基因表达和蛋白质水平的公共数据库。候选基因/蛋白质将依次进行验证，首先通过数据挖掘和信息学来识别合理的机制并与其他表达数据进行比较，然后通过免疫组织化学或定量PCR进行生物化学验证。有限数量的最有趣的基因将在假设检验的第二阶段进行更深入的研究。表达将在不表达基因的细胞系中被强制表达，或者在那些表达基因的细胞系中被敲低。“交付物”将是一组基因和蛋白质，它们在帮助研究正常和恶性组织中上皮-基质相互作用方面的作用经过了几个层次的验证。

DESCRIPTION (provided by applicant): The urothelium lines the bladder and protects it from bacteria and toxic or carcinogenic substances in the urine by maintaining an impermeable barrier. Although this functions remarkably well, it fails in bladder cancer, interstitial cystitis, and possibly other diseases as well. Understanding how the urothelium grows and differentiates and carries out its function may provide important clues to the diagnosis and treatment of bladder diseases. Although urothelium can be cultured, most models fail to adequately model the interactions between the urothelium and the underlying stroma. Together the two form a complex and interacting system. This proposal describes studies to meet the requirements of a PAR to develop cell-selective tools, specifically item (8) identification of cell specific markers to aid studies of epithelial-stromal interactions in normal and malignant tissues and secondarily item (5) discovery of biomarkers that indicate health or mass of individual cell types and item (1) discovery of genes selective to individual cell types. Novel methods for growth of urothelium as an artificial tissue in 3-dimensions and for bladder cancers as artificial tumors on both normal and cancer-modified matrix provides a basis for discovering the genes and proteins involved in defining normal and malignant urothelium. In a first discovery phase, microarrays and a novel proteomics approach with 2-D chromatography will be combined with innovative bioinformatics approaches to identify candidate genes and proteins. A public database of gene expression and protein levels will be constructed. Candidate genes/proteins will be validated sequentially, first by data mining and informatics to identify plausible mechanisms and to compare with other expression data, then biochemically by immunohistochemistry or quantitative PCR. A limited number of the most interesting genes will be investigated in greater depth in a second phase of hypothesis testing. Expression will be forced in cell lines not expressing a gene or knocked down in those that do. The "deliverables" will be sets of genes and proteins subjected to several levels of verification for their role in aiding studies of epithelial-stromal interactions in normal and malignant tissues.