IMAGING BETA CELL FUNCTION WITH BIOSENSORS

描述（由申请人提供）：本提案的总体目标是从生物物理学、生理学和分子生物学的角度了解调节体内β细胞功能的钙（Ca 2+）依赖性刺激-分泌偶联机制。这一目标将通过研究细胞内Ca 2+浓度、线粒体功能、葡萄糖代谢和小鼠和人类胰岛中的胰岛素分泌来实现，其中β细胞已被专门设计用于功能成像。在体外使用完整的胰岛、β细胞的原代培养物和胰岛素瘤细胞已经明确了调节胰岛素分泌的Ca 2+依赖性信号转导机制。尽管在体外方法的重要贡献，在体内完整的胰岛β细胞功能的机制的知识仍然是不完整的。目前成像方法的几个技术限制不允许原位研究胰腺复杂的多细胞环境中的β细胞功能。在这个建议中描述的实验的中心焦点是开发和表征一种新的成像方法，这将有助于原位胰岛细胞功能的研究。在具体目标1中，将使用病毒基因转移载体将具有遗传靶向生物合成荧光Ca 2+传感器的小鼠细胞转染。将评估它们在研究促分泌素刺激后a细胞生物物理和生理反应中的有效性。在特定目标2中，将评估这些传感器作为完整小鼠胰岛中的功能成像指标的效用。在特定目标3中，将对完整人类胰岛内的β细胞进行工程改造，以表达Ca 2+生物传感器，并与小鼠胰岛进行体外研究。在具体目标4中，将开发转基因小鼠模型，其中β细胞已被遗传增强以表达Ca 2+生物传感器，并通过共聚焦显微荧光测定法和胰岛素分泌测量法在体外和体内进行表征。这些研究将为胰岛细胞生物学提供新的认识，这将有利于临床策略，以保护和维持功能性β细胞群。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to understand the calcium (Ca2+)-dependent stimulus-secretion coupling mechanisms that regulate beta-cell function in vivo from the perspectives of biophysics, physiology and molecular biology. This goal will be achieved by studying intracellular Ca 2+ concentration, mitochondrial function, glucose metabolism, and insulin secretion in mouse and human islets in which the beta-cells have been engineered specifically for functional imaging. Ca2+-dependent signal transduction mechanisms that regulate insulin secretion have been well-defined in vitro using intact islets, primary cultures of beta-cells, and insulinoma cells. Notwithstanding the important contributions of the in vitro approaches, knowledge of the mechanisms underlying beta-cell function in intact islets in vivo remains incomplete. Several technical limitations of the current imaging methodologies do not permit the study of beta-cell function within the complex multicellular environment of the pancreas in situ. The central focus of the experiments described in this proposal is to develop and characterize a novel imaging approach, which will facilitate the study of islet cell function in situ. In Specific Aim 1, viral gene transfer vectors will be used to transduce mouse cells with genetically targeted biosynthetic fluorescent Ca 2+ sensors. Their effectiveness in studying a-cell biophysical and physiological responses following secretagogue stimulation will be evaluated. In Specific Aim 2, the utility of these sensors as functional imaging indicators in intact mouse islets will be assessed. In Specific Aim 3, beta-cells within intact human islets will be engineered to express Ca 2+ biosensors and human islet function studied in vitro with comparison to mouse islets. In Specific Aim 4, transgenic mouse models in which the beta-cells have been genetically enhanced to express Ca 2+ biosensors will be developed and characterized in vitro and in vivo by confocal microfluorometry and by measurements of insulin secretion. The studies will provide new understanding of islet cell biology that will benefit clinical strategies to preserve and maintain functional beta-cell mass.