Genes & Proteins of Urothelial-ECM Interaction

描述(申请人提供)：尿路上皮覆盖膀胱，并通过保持不透水屏障来保护其免受细菌和尿液中有毒或致癌物质的侵害。虽然它的作用非常好，但它在膀胱癌、间质性膀胱炎，以及可能还有其他疾病上都失败了。了解尿路上皮是如何生长、分化和执行其功能的，可能会为膀胱疾病的诊断和治疗提供重要线索。虽然尿路上皮可以培养，但大多数模型不能充分模拟尿路上皮和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。这项建议描述了为满足PAR的要求而开展的研究，以开发细胞选择工具，具体地说，项目(8)确定细胞特异性标记物，以帮助研究正常组织和恶性肿瘤组织中的上皮-间质相互作用，其次是项目(5)发现指示单个细胞类型的健康或质量的生物标记物，以及项目(1)发现对单个细胞类型具有选择性的基因。尿路上皮作为人工组织的三维生长和膀胱癌作为人工肿瘤在正常和癌症修饰的基质上生长的新方法为发现与界定正常和恶性尿路上皮有关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创新的生物信息学方法将结合在一起，以识别候选基因和蛋白质。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/蛋白质将被顺序验证，首先通过数据挖掘和信息学来确定可信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进行生化验证。在假设检验的第二阶段，将对数量有限的最有趣的基因进行更深入的研究。在不表达基因的细胞系中，表达将被强迫，在表达基因的细胞系中，表达将被抑制。“可交付成果”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的上皮-间质相互作用方面所起的作用，将接受多个水平的验证。

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.