CORE--CELL PHYSIOLOGY RESOURCE

核心--细胞生理资源

• 批准号: 7069753
• 负责人: Phillip Darwin Bell
• 金额: $ 15.72万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7069753
• 关键词: autosomal recessive trait; biomedical facility; cell biology; cell line; confocal scanning microscopy; disease /disorder model; epithelium; fluorescence; immunofluorescence technique; isolation perfusion; kidney; laboratory mouse; molecular /cellular imaging; pathologic process; physiology; polycystic kidney; renal tubule; tissue /cell culture; tissue /cell preparation; voltage /patch clamp

Genetically engineered mouse models of autosomal recessive polycystic kidney disease (ARPKD) provide model systems to study cyst development in kidney, liver and pancreas. This work has led to the surprising observation that apical cilia play a pivotal role in the pathogenesis of ARPKD. However, there is a lack of information concerning the physiological mechanisms that result in cyst formation. This Cellular Physiology Core will provide a comprehensive facility to perform state-of-the-art physiological experiments in cells and tissues from mouse models of ARPKD. One unique aspect of this Core is the breadth of experimental tools and resources that have been amassed to attack specific problems and questions. The other unique aspect of this Core is the ability to isolate renal tubules and ducts, to produce primary and immortalized cell lines, and to apply experimental procedures and techniques that maximize the physiological information that can be obtained from freshly isolated tissues or cultured cells. The Physiology Core is capable of assessing alterations in the rate of electrolyte and water transport in isolated perfused renal tubules and ductal epithelium and in cultured cells grown on permeable supports. In addition, this Core utilizes advanced fluorescence imaging and multi-photon confocal microscopy to study transporter and channel function, transport rates, and regulation of cytosolic calcium, pH, sodium and chloride. Furthermore, using 2-photon microscopy, this Core now has the ability to visualize and resolve subcellular domains in living cells providing novel and detailed information on ion concentrations in cellular microdomains. For instance, recent work in the Core laboratory has revealed elevated subapical membrane calcium levels in an ARPKD collecting duct cell model. Other capabilities include patch clamp analysis, advanced immunofluorescence techniques, and evaluation of paracrine and autocrine signaling using a biosensor approach. Mice that are generated by the Engineered Mouse Core will be used to obtain fresh tissue and to establish primary and immortalized cell lines. The ability to compare the results obtained in freshly isolated tissues versus primary and immortalized cell lines is one of the major strengths of this Core facility. Overall the Cellular Physiology Core will provide the RPKDCC Investigators with a wide range of tools and expertise to define the physiological mechanisms that are involved in the pathogenesis of ARPKD in renal tubules and epithelial cells.

常染色体隐性多囊肾病 (ARPKD) 基因工程小鼠模型为研究肾脏、肝脏和胰腺囊肿发育提供了模型系统。这项工作令人惊讶地发现，顶端纤毛在 ARPKD 的发病机制中发挥着关键作用。然而，缺乏有关导致囊肿形成的生理机制的信息。该细胞生理学核心将提供综合设施，用于在 ARPKD 小鼠模型的细胞和组织中进行最先进的生理实验。该核心的一个独特之处是为解决特定问题而积累的实验工具和资源的广度。该核心的另一个独特之处是能够分离肾小管和导管，产生原代和永生化细胞系，并应用实验程序和技术，最大限度地从新鲜分离的组织或培养细胞中获得生理信息。生理学核心能够评估电解质和水传输速率的变化 分离的灌注肾小管和导管上皮以及在可渗透支持物上生长的培养细胞中。此外，该核心利用先进的荧光成像和多光子共聚焦显微镜来研究转运蛋白和通道功能、转运速率以及细胞质钙、pH、钠和氯的调节。此外，通过使用 2 光子显微镜，该核心现在能够可视化和解析活细胞中的亚细胞域，提供有关细胞微域中离子浓度的新颖且详细的信息。例如，核心实验室最近的工作揭示了 ARPKD 集合管细胞模型中心尖下膜钙水平升高。其他功能包括膜片钳分析、先进的免疫荧光技术以及使用生物传感器方法评估旁分泌和自分泌信号传导。由工程小鼠核心产生的小鼠将用于获取新鲜组织并建立原代和永生化细胞系。能够将新鲜分离的组织与原代细胞系和永生化细胞系获得的结果进行比较，这是该核心设施的主要优势之一。总体而言，细胞生理学核心将为 RPKDCC 研究人员提供广泛的工具和专业知识，以定义肾小管和上皮细胞中 ARPKD 发病机制所涉及的生理机制。