Inter-islet insulin dynamics measured via controlled microfluidic stimulation

描述（由申请人提供）：本研究的目的是开发和利用新型微流体装置，结合现有的成像方法，以阐明完整胰岛的细胞间信号动力学。关于胰腺中多个胰岛协调振荡的可能机制知之甚少，但在整个动物和人类胰腺中观察到的全球脉动分泌表明，这些胰岛之间存在明显的协调。由于这种搏动行为在糖尿病患者中被破坏，阐明这些机制可以为糖尿病病情提供相当大的见解。在这个提议中，假设旁分泌信号在协调多个胰岛之间的振荡行为中起主要作用。典型的胰岛直径在100-200微米的范围内，大多数常用的微流体装置中的通道尺寸被证明是分离和研究这些特定细胞的理想选择。因此，微流体工具将通过创建可以使用片上阀打开或关闭的胰岛间通信网络来研究这种协调。在Specific Aim 1中，将优化微流体结构、流量控制方法和细胞操作策略，以将锌离子浓度与单个活胰岛的胰岛素分泌联系起来。申请人先前开发的非脉动流量控制方法将用于刺激浸泡在锌指示剂染料中的胰岛，以实现单个胰岛胰岛素分泌的定量实时成像。在特异性目标2中，旁分泌信号在多个活胰岛细胞间振荡行为中的作用将被确定。在Specific Aim 1中开发的新型微流体工具将用于通过精确控制的非脉冲刺激将多个胰岛定位到通信网络中。这些实验将比较正常胰岛、药理学改变的正常胰岛和基因操纵的3IRKO小鼠的胰岛之间的交流，这些小鼠失去了胰岛素分泌活性，类似于II型糖尿病小鼠的胰岛。这个项目是专门设计的，通过使用申请人在微观分析方法方面的专业知识，提供定量成像和生物方法方面的培训。理解这些类型的胰岛行为的机制途径的重要性不能被夸大。在美国，糖尿病影响着2080万儿童和成人（占人口的7.0%），2002年估计花费1320亿美元；这种疾病会导致严重的并发症，如心脏病、中风、失明、肾病和神经系统疾病。

DESCRIPTION (provided by applicant): The objective of this proposed research is to develop and utilize novel microfluidic devices in concert with established imaging methods in order to elucidate intercellular signaling dynamics among intact pancreatic islets of Langerhans. Little is known about possible mechanisms that coordinate oscillations throughout multiple islets in the pancreas, but the globally pulsatile secretion that has been observed in the pancreas of whole animals and humans suggests an obvious coordination among these islets. Since this pulsatile behavior is disrupted in diabetic patients, the elucidation of these mechanisms could provide considerable insight into the diabetic condition. In this proposal, it is hypothesized that paracrine signaling plays a major role in coordinating oscillatory behavior among multiple islets. With the typical islet diameter in the range of 100-200 micrometers, the channel dimensions in most commonly used microfluidic devices prove to be ideal for isolating and studying these particular cells. Thus, microfluidic tools will be utilized to investigate this coordination by creating inter-islet communication networks that can be switched on or off using on-chip valves. In Specific Aim 1, microfluidic architectures, flow control methods, and cell manipulation strategies will be optimized to correlate zinc ion concentration with insulin secretion from single live islets. Non- pulsatile flow control methods previously developed by the applicant will be used to stimulate islets bathed in zinc-indicator dye to allow quantitative, real-time imaging of insulin secretion from individual islets. In Specific Aim 2, the role of paracrine signaling in intercellular oscillatory behavior among multiple live islets will be determined. The novel microfluidic tools developed in Specific Aim 1 will be used to position multiple islets into communication networks with precisely controlled, non-pulsatile stimulation. These experiments will compare communication among normal islets, pharmacologically altered normal islets, and islets from genetically manipulated (3IRKO mice, which have lost insulin secretory activity, resembling islets from mice with Type II diabetes. This project is specially designed to provide training in quantitative imaging and biological methods through use of the applicant's expertise in micro-analytical methodology. The importance of understanding these types of mechanistic pathways of islet behavior can not be overstated. Diabetes affects 20.8 million children and adults in the United States (7.0% of population), and cost an estimated $132 billion in 2002; the disorder can result in serious complications such as heart disease, stroke, blindness, kidney disease, and nervous system disease.