IMAGING BETA CELL FUNCTION WITH BIOSENSORS

这项建议的总体目标是理解依赖CAZ*的刺激-分泌耦合 从生物物理学、生理学和生物学角度研究体内13细胞功能的调控机制 分子生物学。这一目标将通过研究细胞内钙浓度、线粒体 13细胞所在的小鼠和人胰岛的功能、葡萄糖代谢和胰岛素分泌 是专门为功能成像设计的。钙依赖的信号转导机制 在体外使用完整的胰岛，原代培养的13个细胞，已经很好地定义了调节胰岛素分泌的功能。 细胞和胰岛素瘤细胞。尽管体外方法有重要贡献， 对体内完整胰岛13细胞功能的潜在机制的了解仍然不完整。 当前成像方法的几个技术限制不允许对13-细胞进行研究 在复杂的胰腺多细胞环境中原位发挥作用。会议的中心焦点是 本提案中描述的实验旨在开发和表征一种新的成像方法，该方法 为原位研究胰岛细胞功能奠定了基础。在特定目标1中，病毒基因转移载体将是 用于用基因靶向的生物合成荧光钙传感器转导小鼠细胞。他们的 促分泌剂后Q细胞生物物理和生理反应研究的有效性 将对刺激进行评估。在具体目标2中，这些传感器作为功能成像的用途 将对完整的小鼠胰岛的指标进行评估。在特定的目标3，13-完整的人类胰岛内的细胞将 基因工程表达钙生物传感器和人胰岛功能的体外研究与比较 小鼠的小岛。在特定目标4中，转基因小鼠模型中的13-细胞已被遗传 增强表达钙离子的生物传感器将通过以下方法在体外和体内得到开发和表征 通过共聚焦显微荧光法和胰岛素分泌的测量。这些研究将提供新的 对胰岛细胞生物学的了解将有助于临床策略保存和维持功能 13个细胞团。

The overall goal of this proposal is to understand the CaZ*-dependent stimulus-secretion coupling mechanisms that regulate 13-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 13-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 13- cells, and insulinoma cells. Notwithstanding the important contributions of the in vitro approaches, knowledge of the mechanisms underlying 13-cell function in intact islets in vivo remains incomplete. Several technical limitations of the current imaging methodologies do not permit the study of 13-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 Q-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, 13-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 13-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 13-cell mass.