Microchip-based Cell Reactor/Analysis System

基于微芯片的细胞反应器/分析系统

• 批准号: 7363891
• 负责人: ROBERT Scott MARTIN
• 金额: $ 22.05万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-10 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7363891
• 关键词: Affect; Age; Calcium; Carbon; Catecholamines; Cell Culture System; Cell Line; Cell model; Cells; Cerebrospinal Fluid; Characteristics; Chronic; Computer Systems Development; Condition; Coupled; Cultured Cells; Detection; Development; Devices; Dimensions; Disease; Dopamine; Dopaminergic Cell; Electrodes; Electrophoresis; Funding; Goals; Grant; Immobilization; In Vitro; Incidence; Incubated; Inflammation; Ink; Measures; Mediating; Microchip Electrophoresis; Microglia; Mitochondria; Modeling; Molecular; Monitor; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Neurotransmitters; Nitric Oxide; Nitrites; Norepinephrine; Numbers; Parkinson Disease; Patients; Physicians; Play; Procedures; Production; Protocols documentation; Publishing; Reporting; Role; Sampling; Scheme; Solutions; Source; Substantia nigra structure; System; Systems Analysis; Technology; Thinking; United States; Vesicle; Work; analytical tool; base; dopaminergic neuron; human NOS2A protein; in vivo; microchip; neurotransmitter release; oxidation; size; synergism; trait; voltage

DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop a microchip-based system that integrates a cell culture model with a separation-based analysis system to study the affect of nitric oxide (NO) on dopaminergic degeneration. Degeneration of dopaminergic neurons is a characteristic of Parkinson's disease and studies have shown that inflammation of microglia results in the activation of inducible nitric oxide synthase, which produces NO that may enter the dopaminergic cells and oxidize dopamine found in storage vesicles. This NO-mediated oxidation may render dopamine stores depleted, thus decreasing amounts of exocytotic dopamine, a known trait of Parkinson's patients. Given all that is known about intracellular neuronal changes that may be related to the genesis of Parkinson's disease, a detailed molecular level understanding of the mechanisms leading to dopaminergic degeneration by NO is still lacking. An optimal situation to study these mechanisms is to employ an in vitro cell culture system that mimics an in vivo system and is easily coupled to an analysis system that can discretely analyze the products formed when NO depletes dopamine stores. In this proposal, we describe the development of such a system using microchip technology and PC 12 cells. Great strides towards the development of this system were made in the currently funded grant. The proposed studies will continue the development of a microchip-based cell culture system/analysis system that incorporates PC 12 cell immobilization, on-chip valving, electrophoresis-based separations, and electrochemical detection. To accomplish this, we first will integrate PC 12 cell immobilization and amperometric detection with the continuous flow reactor/microchip electrophoresis device developed in the current studies. Variables such as immobilization conditions and chip dimensions will be optimized so that the cells become nearly confluent in rounded microchannels. We then propose to use this device to study the effect of NO on dopaminergic degeneration by monitoring changes in dopamine and norepinephrine release as the cells are incubated with varying concentrations of NO. We also propose to determine the different products that result from the interaction of NO with these neurotransmitters. Finally, we propose to develop a more realistic model of Parkinson's onset by culturing both PC 12 cells and microglia cells in a microchip device, with the long term goal of using the microglia as the NO source in these studies. This will involve optimization of immobilization conditions for the microglia as well as studies to show that the microglia cells are bioresponsive. While this proposal is focused on PC 12 cells and neurotransmitter analysis, the final device will be a general analytical tool that is amenable to immobilization of a variety of cell lines and analysis of released analytes by electrophoretic means. The overall goal of this proposal is to develop a microchip device that can be used to study the role nitric oxide plays in dopaminergic degeneration, which is a characteristic of Parkinson's disease. This final device will utilize micron-sized valves to integrate a neuron cell model to an analysis system that can differentiate between the neurotransmitters released from the cells. This device will allow neuroscientists to study the role that microglia cells play in the onset of Parkinson's disease.

描述（由申请人提供）：本提案的总体目标是开发一种基于微芯片的系统，该系统将细胞培养模型与基于分离的分析系统相结合，以研究一氧化氮（NO）对多巴胺能变性的影响。多巴胺能神经元的变性是帕金森病的特征，并且研究已经表明，小胶质细胞的炎症导致诱导型一氧化氮合酶的激活，其产生可以进入多巴胺能细胞并氧化在储存囊泡中发现的多巴胺的NO。这种NO介导的氧化可能使多巴胺储存耗尽，从而减少胞吐多巴胺的量，这是帕金森病患者的已知特征。鉴于所有已知的细胞内神经元的变化，可能与帕金森氏病的发生，一个详细的分子水平的理解，导致多巴胺能变性的机制，NO仍然缺乏。研究这些机制的最佳情况是采用体外细胞培养系统，该系统模拟体内系统，并且易于与分析系统偶联，该分析系统可以离散分析NO耗尽多巴胺储存时形成的产物。在这个建议中，我们描述了这样一个系统的发展，使用微芯片技术和PC 12细胞。在目前资助的赠款方面，这一系统的发展取得了很大进展。拟议的研究将继续开发基于微芯片的细胞培养系统/分析系统，该系统包括PC 12细胞固定化、芯片上阀门、基于荧光素酶的分离和电化学检测。为了实现这一点，我们首先将PC 12细胞固定化和安培检测与目前研究中开发的连续流动反应器/微芯片电泳装置相结合。将优化诸如固定化条件和芯片尺寸的变量，使得细胞在圆形微通道中变得几乎汇合。然后，我们建议使用这个设备来研究NO对多巴胺能变性的影响，通过监测多巴胺和去甲肾上腺素释放的变化，细胞与不同浓度的NO孵育。我们还建议确定不同的产品，结果从NO与这些神经递质的相互作用。最后，我们建议通过在微芯片装置中培养PC 12细胞和小胶质细胞来开发更现实的帕金森病发作模型，长期目标是在这些研究中使用小胶质细胞作为NO来源。这将涉及优化小胶质细胞的固定条件以及研究表明小胶质细胞是生物反应性的。虽然该提议集中于PC 12细胞和神经递质分析，但最终的装置将是一种通用的分析工具，其适合于固定各种细胞系和通过电泳手段分析释放的分析物。 该提案的总体目标是开发一种微芯片设备，可用于研究一氧化氮在多巴胺能变性中的作用，这是帕金森病的一个特征。最终的设备将利用微米大小的阀门将神经元细胞模型集成到分析系统中，该分析系统可以区分从细胞释放的神经递质。该设备将使神经科学家能够研究小胶质细胞在帕金森病发病中的作用。