BILIARY SECRETORY PATHWAYS IN CYSTIC FIBROSIS

囊性纤维化中的胆道分泌途径

• 批准号: 6342402
• 负责人: WON KYOO CHO
• 金额: $ 7.14万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6342402
• 关键词: adenosine triphosphate; bicarbonates; bile ducts; biological signal transduction; bombesin; chloride channels; cystic fibrosis; disease /disorder model; ion transport; laboratory mouse; micropuncture; microspectrophotometry; neuropeptides; secretion; tissue /cell culture; ursodeoxycholate; vasoactive intestinal peptide; video microscopy; voltage /patch clamp

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.

囊性纤维化是白人人群中最常见的致死性遗传病。随着慢性肝病患者寿命的延长，肝病已成为第二大死亡原因。CF病的发生是由于胆管分泌缺陷导致顽固的胆汁分泌物阻塞胆管，从而在局限性门脉周围胆管纤维化/肝硬变中分泌。这一解释，再加上最近发现CFTR只在胆管细胞上表达，而不是在肝细胞上表达，表明研究胆汁分泌对于了解CF肝的病理生理机制和制定治疗策略至关重要。从大鼠肝脏制备的极化分离胆管单位(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鉴定正常和CF基因敲除小鼠的胆管细胞(BDC)和IBDU，鉴定BDC中的离子转运体，并研究包括神经内分泌肽在内的各种促分泌剂在胆汁分泌中的作用和机制，以期找到激活CF小鼠胆汁分泌途径的途径。从正常小鼠分离IBDU的初步实验产生了对分泌素、血管活性肠肽和DBcAMP-IBMX有反应的完整的极化功能IBDU。类似的IBDU也从CF小鼠中分离出来，但需要进一步鉴定。定量视频显微镜将用于筛选潜在的促分泌剂，以刺激正常和CF小鼠的胆汁分泌，并通过离子替代和抑制剂研究来表征其潜在的离子转运体。这些离子转运体将通过BCECF双比值法测量pH、微穿孔和膜片钳技术进行进一步研究。将通过监测次级信使浓度的变化来研究参与其作用的信号转导系统。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克服CFTR缺陷的治疗方法。反过来，该项目将为候选人提供一个极好的机会来扩大和发展研究和认知技能，使其成为独立的研究人员，并有助于成功竞争未来的研究拨款。