BILIARY SECRETORY PATHWAYS IN CYSTIC FIBROSIS

囊性纤维化（CF）是白色人群中最常见的致死性遗传病。随着CF患者寿命的延长，肝病已成为第二大死亡原因。CF疾病的发展被认为是由于胆管中的分泌缺陷导致胆管被顽固的胆汁分泌物阻塞，从而在局灶性门静脉周围胆汁纤维化/肝硬化中分泌。除了最近发现CFTR仅在胆管细胞上表达而不在肝细胞上表达之外，这种解释表明研究胆汁分泌对于理解CF肝脏的病理生理学和开发治疗策略至关重要。从大鼠肝脏制备的一种新型极化离体胆管单位（IBDU）已被证明是研究胆管分泌的理想工具，但缺乏CF大鼠模型限制了其在CF研究中的应用。通过应用这些分离方法，最近，已经从正常和CF小鼠中分离出IBDU。因此，本研究的目的是进一步表征正常和CF基因敲除小鼠的胆管细胞（BDC）和IBDU，表征BDC中的离子转运蛋白，并研究包括神经内分泌肽在内的各种促分泌素在胆汁分泌中的作用和机制，以找到激活CF小鼠中替代的、cAMP非依赖性胆汁分泌途径的方法。从正常小鼠中分离IBDU的初步实验产生了对促胰液素、血管活性肠肽和DBcAMP-IBMX有反应的完整极化功能性IBDU。类似的IBDU也从CF小鼠中分离，但需要进一步表征。定量视频显微镜将用于筛选刺激正常和CF小鼠胆汁分泌的潜在促分泌素，并通过使用离子置换和抑制剂研究表征其基础离子转运蛋白。这些离子转运蛋白将进一步研究BCECF双比率法测量pH值，微穿刺，膜片钳技术。参与其行动的信号转导系统将通过监测第二信使浓度的变化进行研究。了解正常和CF小鼠胆汁分泌的转运系统及其潜在机制将有助于制定治疗方法来克服CFTR缺陷。反过来，该项目将为候选人提供一个极好的机会，以扩大和发展研究和认知技能，成为独立的研究人员，以及帮助成功地竞争未来的研究赠款。

Cystic fibrosis (CF) is the most common lethal inherited diseases in white population. As CF patients live longer, liver disease has become the second leading cause of death. The development of CF disease is believed to result from the secretory defects in the bile ducts leading to the obstructions of bile ductules by tenacious bile secretions, thereby secreting in focal periportal biliary fibrosis/cirrhosis. This explanation in addition to the recent finding that CFTR is only expressed on bile duct cells, but not on hepatocytes, suggest that studying biliary secretion is crucial to understanding the pathophysiology and developing therapeutic strategies for CF liver. A novel polarized isolated bile duct unit (IBDU) prepared from rat liver has demonstrated to be an ideal tool to study bile ductular secretion but the lack of CF rat model limited its use in CF studies. By applying these isolation methods, recently, IBDUs have been isolated from normal and CF mice. Therefore, the aims of this research are to further characterize bile duct cells (BDC) and IBDU from normal and CF knockout mice, to characterize ion transporters in BDC, and to study the actions and mechanisms of various secretagogues including neuroendocrine peptides in biliary secretion in order to find ways to activate alternative, cAMP-independent biliary secretory pathways in CF mice. Preliminary experiments to isolate IBDU from normal mouse yielded intact polarized functional IBDU that responds to secretin, vasoactive intestinal peptide, and DBcAMP-IBMX. Similar IBDUs were also isolated from CF mice but need further characterization. Quantitative videomicroscopy will be used to screen potential secretagogues to stimulate biliary secretion in normal and CF mice and to characterize their underlying ion transporters by using ion substitutions and inhibitor studies. These ion transporters will be further studied by BCECF dual ratio methods for measuring pH, micropuncture, and patch clamping techniques. Signal transduction systems involved in their action will be studied by monitoring changes in the concentrations of secondary messengers. Understanding transport systems and their underlying mechanisms of biliary secretion in normal and CF mice will help to formulate therapeutic approaches to overcome the CFTR defect. This project, in turn, will provide the candidate with an excellent opportunity to broaden and develop research and cognitive skills to become independent researcher, as well as help to successfully compete for future research grants.