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DEVELOPMENT OF NOVEL PANCREATIC BETA CELL MODELS

新型胰腺β细胞模型的开发

基本信息

批准号：
2906374
负责人：
PATRICIA M. HINKLE
金额：
$ 14.91万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-09-30 至 2001-09-29
项目状态：
已结题

项目摘要

DESCRIPTION (taken from the application) Insulin production in the islets of Langerhans is regulated by Multiple hormonal, neuronal and paracrine factors acting through interconnected signal transduction pathways. The intracellular concentration of free Ca2+ in the cytoplasm is an important determinant of insulin secretion. We propose that the concentration of Ca2+ in the endoplasmic reticulum (ER) is also important in regulation of the pancreatic beta cell, and that the distinct patterns of Ca2+ responses evoked by glucose and other stimuli are translated differentially into down stream responses. We will develop novel methods to determine how regulators of insulin production affect Ca2+ concentrations in the ER and how changes in cellular Ca2+ levels are translated into a prototypic response, activation of calmodulin. Two classes of fluorescent indicators, both containing two spectral variants of green fluorescent protein, will be expressed in beta cells and fluorescence resonance energy transfer (FRET) will be measured by fluorescence microscopy. One set of reporters, the cameleons, shows changes in FRET upon Ca2+ calmodulin binding. We will transfect MIN6 cells with Ca2+ sensing cameleons targeted to the ER, YCer3 and Ycer4, and determine: (1) how the concentration of Ca2+ in the ER changes in response to activation of a G protein-coupled receptor linked to phospholipase C, (2) whether high glucose release Ca2+ from the ER when it is added alone, with carbachol or with vasoactive intestinal peptide, and (3) whether changes in ER Ca2+ are restricted to certain regions of the ER. We will also introduce the fluorescence target FIP-CBsm into MIN6 cells and use FRET to measure the extent of Ca2+-calmodulin binding. We will use this model to determine the spatial and temporal patterns of Ca2+-calmodulin activation when beta cells are: (1) exposed to stimuli that cause release of Ca2+ from intracellular stores and those that cause Ca2+ influx through voltage-operated Ca2+ channels, and 2) exposed to stimuli that cause sustained versus oscillatory increases in the cytoplasmic free Ca2+ concentration.

描述(取自应用程序) 朗格汉斯胰岛胰岛素的产生受多种因素的调节激素、神经元和旁分泌因子通过相互关联发挥作用信号转导通路。细胞内游离钙离子浓度在细胞质中是胰岛素分泌的重要决定因素。我们提出内质网内钙离子浓度的变化在调节胰岛β细胞方面也很重要，而且葡萄糖和其他刺激引起的钙离子反应的不同模式以不同方式转化为下游响应。我们将发展确定胰岛素生成调节剂如何影响的新方法内质网中的钙离子浓度以及细胞内钙离子水平的变化转化为典型的反应，激活钙调蛋白。二两类荧光指示剂，均包含两种光谱变体绿色荧光蛋白，将在β细胞中表达，荧光共振能量转移(FRET)将通过以下方法测量荧光显微镜。一组记者，骆驼，展示了钙调素结合时FRET的变化。我们将转染MIN6细胞以ER、YCer3和Ycer4为靶点的钙离子感应卡梅隆，以及测定：(1)内质网内钙离子浓度如何变化激活与磷脂酶C相连的G蛋白偶联受体， (2)高糖单独加入时是否从内质网释放钙离子，卡巴胆碱或血管活性肠肽，以及(3)是否内质网钙离子的变化仅限于内质网的某些区域。我们会还将荧光靶标FIP-CBSM导入MIN6细胞并使用 FRET检测钙-钙调蛋白结合的程度。我们将使用这个确定钙离子-钙调蛋白时空模式的模型 β细胞在以下情况下的激活：(1)暴露于导致释放的刺激从细胞内存储的钙离子和那些引起钙离子内流的电压操作的钙离子通道，以及2)暴露于导致胞浆内游离钙离子的持续性与振荡性升高集中精神。