IMAGING BETA CELL FUNCTION WITH BIOSENSORS

描述(申请人提供)：本方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(Ca~(2+))依赖的刺激-分泌耦合机制。这一目标将通过研究小鼠和人类胰岛的细胞内钙浓度、线粒体功能、葡萄糖代谢和胰岛素分泌来实现，在这些胰岛中，β细胞已被专门设计用于功能成像。在体外，利用完整的胰岛、原代培养的β细胞和胰岛素瘤细胞，已经很好地确定了调节胰岛素分泌的依赖于钙的信号转导机制。尽管体外方法做出了重要贡献，但对活体完整胰岛中潜在的β细胞功能的机制的了解仍然不完整。目前成像方法的几个技术限制不允许在原位研究胰腺复杂的多细胞环境中的β细胞功能。这项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，这将有助于原位研究胰岛细胞的功能。在具体目标1中，病毒基因转移载体将被用来转导具有基因靶向的生物合成荧光钙传感器的小鼠细胞。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。在具体目标2中，将评估这些传感器作为完整小鼠胰岛的功能成像指示器的用途。在特定的目标3中，完整的人胰岛中的β细胞将被改造成表达钙生物传感器，并在体外研究人的胰岛功能，并与小鼠胰岛进行比较。在特定的目标4中，将建立转基因小鼠模型，在该模型中，β细胞被遗传增强以表达钙生物传感器，并通过共聚焦显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理解，这将有助于临床策略保存和维持功能性β细胞团。

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.