Alterations of the expression pattern in bile-duct epithelia and the bile-proteome in primary sclerosing cholangitis: Identification of disease-associated proteins by a proteomic approach

胆管细胞癌起源于胆管上皮细胞。它与非常差的预后有关。CCC最重要的危险因素是原发性硬化性胆管炎(PSC)，这是胆管的一种慢性进行性炎症。在赠款支持的第一阶段，建立了必要的方法学，然后检测了胆汁蛋白的疾病特异性差异，并确定了候选蛋白。在这项拨款申请中，我们的目标是验证和描述这些候选人。此外，我们的目标是确定胆汁蛋白质组的炎症-异型增生/癌的序列，这将使我们能够对PSC患者进行风险分层。基于已有的PSC相关基因型别的数据，我们希望确定胆汁蛋白质组的特定基因型差异。在第一次赠款支持期间收集的胆汁样本和可用的临床信息将被使用，以及持续收集的新样本。我们将使用已建立的比较蛋白质组学方法(二维荧光差示凝胶电泳和质谱仪)。确定的候选蛋白质将使用炎症细胞培养模型进行验证和表征。作为项目的继续，我们将进行胆管活检和刷状细胞学的基因表达谱分析。这些试验的主要内容将是在赠款支助的第一阶段确定的方法和获得的数据。此外，我们希望通过对胆汁样本的分离来加强对胆汁蛋白质组的分析。我们最感兴趣的是从胆汁中分离和鉴定外切体。作为初步研究工作的一部分，这些胆汁外切体已经被检测到。此外，我们还想分析不同疾病组之间胆汁外切体的蛋白质组成，这可能导致在外切体中检测疾病特异性标记蛋白。所有这些数据都应该在分组之间进行分析，并考虑到临床参数。基于我们的研究，我们可能会对PSC相关性胆管细胞癌的早期诊断以及治疗PSC的新的治疗方法获得新的见解。此外，我们也许能够开发出一种描述慢性炎症向不典型增生和癌症转变的癌症序贯发展模型。

Cholangiocellular carcinoma (CCC) originates from biliary epithelial cells. It is associated with a very poor prognosis. The most important risk factor for CCC is primary sclerosing cholangitis (PSC), a chronic progressive inflammation of the bile ducts. In the first period of grant support the necessary methodology was established and afterwards disease specific differences of the biliary proteins were detected and candidate proteins were identified. In this grant application we aim at verifying and characterizing these candidates. Furthermore we aim to identify a sequence of inflammation-dysplasia/carcinoma of the bile proteome that would allow us to perform risk stratification of patients with PSC. Based on the already available data on PSC-associated genotypes we would like to identify genotype specific differences of the bile proteome.Bile samples that have been collected during the first period of grant support and the available clinical information will be used as well as new samples that are continuously collected. We will use the established methodology of comparative proteomics (2-D- Fluorescence Difference Gel Electrophoresis and mass spectrometry). The identified candidate proteins will be verified and characterized using cell culture models of inflammation. As continuation of the project we will perform gene expression profiles of biopsies from the bile duct and brush cytologies. Mainstay for these experiments will be the methodology established and data acquired during the first period of grant support. Moreover we would like to augment the analysis of the bile proteome by fractionation of the bile samples. Of main interest we would like to isolate and characterize exosomes from bile. These biliary exosomes have already been detected as part of preliminary research work. In addition we would like to analyze the protein composition of biliary exosomes between different disease groups which might lead to the detection of disease specific marker proteins in exosomes. All these data should be analyzed between groups and taking into account clinical parameters. The necessary bioinformatics tools are available.Based on our study we might acquire new insights into early diagnosis of PSC associated cholangiocarcinoma as well as new therapeutic approaches for treatment of PSC. Furthermore we might be able to develop a model of a sequential cancer development which describes the transition of chronic inflammation to dysplasia and carcinoma.