Pathobiology of Hepatic Epithelia

描述（由申请人提供）：我们的长期目标仍然是应用细胞和分子生物学的基本概念和广泛技术来了解肝上皮细胞的功能和功能障碍。我们继续关注胆管细胞，肝内胆管上皮细胞，因为它们的生物学和临床重要性，因为我们已经开发了新的概念、假设和技术来研究胆管细胞病理生物学，这是肝脏研究的一个不足的领域。我们实验室最近的证据表明：(i)水通道蛋白（AQPs），一个水通道家族，在胆管形成中很重要；（ii）胆管细胞含有初级纤毛，作为感觉细胞器，参与正常胆汁形成和胆道膀胱形成。因此，我们将检验导管胆汁形成的中心假设：(i)是溶质驱动的水分子被动运动的净结果，水分子通过不同细胞区室中组成性表达的aqp或在不同细胞区室中循环；（ii）受经顶端胆管细胞膜上的初级纤毛感知的腔内机械、化学和渗透信号的影响；（iii）当胆管细胞纤毛结构和/或功能紊乱时，在常染色体隐性遗传性多囊肾病（ARPKD）的遗传自发或实验动物模型中异常。我们的三个不同但综合的特定目标验证了以下假设：(i)胆总管形成涉及每个胆管细胞顶膜上表达的初级纤毛的正常功能，以检测来自胆汁的机械（如胆汁流速），化学（如核苷酸，胆汁酸，葡萄糖）和/或渗透（如胆汁低/高渗）信号；（ii）调节胆管形成的关键“通量”蛋白（如AQPs、AE2、CFTR）的细胞表达、区隔化和再循环受到纤毛刺激的影响；（iii）纤毛相关蛋白的结构、表达和/或细胞定位异常（例如，纤维囊蛋白，PKHD1的蛋白产物，ARPKD中突变的基因）导致胆管细胞水、溶质和离子运输紊乱，促进胆囊炎的发生。我们将采用已建立的和新的方法、模型和探针，包括：灌注胆管单元、分离胆道囊肿、分离胆管细胞纤毛、自发性（即PCK大鼠）和转基因（即纤维囊素敲除小鼠）ARPKD动物模型、使用小干扰rna （sirna）进行基因沉默、新型胆管细胞培养系统和创新的形态学技术。我们的研究结果将进一步阐明AQPs在胆管细胞胆汁形成中的作用，直接解决胆管细胞纤毛在胆管胆汁产生中的潜在重要性，并探讨胆管细胞纤毛与胆道胆囊形成中可能出现的水、离子和溶质运输紊乱的关系。我们项目的创新方面包括新的方法和动物模型，以及关于胆管细胞aqp和纤毛在胆管胆汁形成和胆道膀胱发生中的重要性的新概念。我们将提供信息以获得对正常胆管细胞功能的新见解，探索胆管细胞功能障碍的高度有前途的选择方面，并继续为理解和管理胆管疾病提供广泛的理论框架，胆管疾病是一组遗传性和获得性肝胆疾病，其中胆管细胞是各种病理过程的主要目标。

DESCRIPTION (provided by applicant): Our long-term objectives remain to apply the fundamental concepts and broad technologies of cell and molecular biology to understand hepatic epithelial cell function and dysfunction. We continue to focus on cholangiocytes, the epithelial cells lining intrahepatic bile ducts, because of their biologic and clinical importance, and because of the new concepts, hypotheses, and techniques we have developed to study, cholangiocyte pathobiology, an underserved area of liver research. Recent evidence from our lab indicates that: (i) aquaporins (AQPs), a family of water channels, are important in ductal bile formation; and (ii) cholangiocytes contain primary cilia that act as sensory organelles and participate in normal bile formation and in biliary cystogenesis. Thus, we will test the central hypothesis that ductal bile formation: (i) is the net result of solute-driven, passive movement of water molecules through AQPs constitutively expressed in or recycled among distinct cellular compartments; (ii) is influenced by luminal mechanical, chemical, and osmotic signals sensed via primary cilia on the apical cholangiocyte membrane; and (iii) is abnormal in genetic spontaneous or experimental animal models of autosomal recessive polycystic kidney disease (ARPKD) when cholangiocyte ciliary structure and/or function is disturbed. Our three distinct but integrated specific aims test the hypotheses that: (i) ductal bile formation involves the normal function of primary cilia expressed on the apical membrane of each cholangiocyte to detect mechanical (e.g., bile flow rate), chemical (e.g., nucleotides, bile acids, glucose), and/or osmotic (e.g., bile hypo/hyperosmolarity) signals from bile; (ii) cellular expression, compartmentalization, and recycling of key 'flux' proteins (e.g., AQPs, AE2, CFTR) regulating ductal bile formation are influenced by ciliary stimulation; and (iii) abnormalities in structure, expression, and/or cellular localization of cilia-associated proteins (e.g., fibrocystin, the protein product of PKHD1, the gene mutated in ARPKD) contribute to disturbances in cholangiocyte water, solute, and ion transport promoting biliary cystogenesis. We will employ established and new methods, models, and probes, including: perfused bile duct units, isolated biliary cysts, isolated cholangiocyte cilia, spontaneous (i.e., the PCK rat) and transgenic (i.e., fibrocystin knockout mouse) animal models of ARPKD, gene silencing using small-interfering RNAs (siRNAs), novel cholangiocyte culture systems, and innovative morphologic techniques. Our results will further clarify the role of AQPs in cholangiocyte bile formation, address directly the potential importance of cholangiocyte cilia in ductal bile production, and explore the relationship of cholangiocyte cilia to possible disturbances of water, ion, and solute transport in biliary cystogenesis. Innovative aspects of our program include novel methodologies and animal models, and new concepts regarding the importance of cholangiocyte AQPs and cilia in ductal bile formation and biliary cystogenesis. We will generate information to yield new insights into normal cholangiocyte function, explore highly promising, selected aspects of cholangiocyte dysfunction, and continue to provide a broad theoretical framework for understanding and managing the cholangiopathies, a group of genetic and acquired hepatobiliary diseases in which the cholangiocyte is the principal target of diverse pathologic processes.