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体内调节β细胞功能的钙（Ca2+）依赖性刺激-分泌偶联机制。这一目标将通过研究细胞内ca2 +浓度、线粒体功能、葡萄糖代谢和小鼠和人类胰岛的胰岛素分泌来实现，其中β细胞已被专门用于功能成像。调节胰岛素分泌的Ca2+依赖信号转导机制已经在体外使用完整的胰岛、β细胞的原代培养和胰岛素瘤细胞进行了明确的研究。尽管体外方法做出了重要贡献，但对体内完整胰岛中β细胞功能的机制的了解仍然不完整。当前成像方法的一些技术限制不允许在胰腺原位复杂的多细胞环境中研究β细胞功能。本提案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，这将有助于原位研究胰岛细胞功能。在Specific Aim 1中，病毒基因转移载体将用于转导具有基因靶向生物合成荧光ca2 +传感器的小鼠细胞。它们在研究促分泌剂刺激后的a细胞生物物理和生理反应方面的有效性将被评估。在特异性目标2中，将评估这些传感器作为完整小鼠胰岛功能成像指标的效用。在Specific Aim 3中，完整的人胰岛内的β细胞将被改造成表达ca2 +生物传感器和人胰岛功能，并与小鼠胰岛进行比较。在Specific Aim 4中，将开发转基因小鼠模型，其中β细胞经过基因增强以表达ca2 +生物传感器，并通过共聚焦微荧光法和胰岛素分泌测量在体外和体内进行表征。这些研究将提供对胰岛细胞生物学的新认识，这将有利于临床策略的保护和维持功能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.