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Microchip-based Cell Reactor/Analysis System

基于微芯片的细胞反应器/分析系统

基本信息

批准号：
6848225
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
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-12-10 至 2008-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6848225
关键词：
PC12 cells Parkinson&apos s disease calcium capillary electrophoresis disease /disorder model dopamine exocytosis microglia microprocessor /microchip model design /development neural degeneration nitric oxide nitric oxide synthase norepinephrine oxidation tissue /cell culture

项目摘要

DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop a microchip-based cell culture model that is integrated with a separation-based analysis system to eventually 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 (iNOS), which produces NO that may enter the dopaminergic cells (such as PC 12 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. These findings may lead to a possible mechanism for NO-mediated damage to dopaminergic cells; however, a molecular level understanding of the mechanisms leading to dopaminergic degeneration is lacking. Since understanding these mechanisms at the molecular level is difficult to achieve in vivo, cell culture models are often employed. To gain insight into these mechanisms, we propose to develop a microchip-based system that incorporates PC 12 cells to study the affect of NO on dopaminergic degeneration. To accomplish this, a three-dimensional PC 12 cell culture model will be immobilized on a planar microchip. These cells will be shown to be bioresponsive by monitoring the neurotransmitters (dopamine and norepinephrine) that are released upon stimulation with calcium. To enable the quantitative identification of the neurotransmitters released from the PC 12 cells, a microchip capillary electrophoresis-based separation system will be developed. The system will utilize amperometric detection to monitor dopamine and norepinephrine released by the cells upon stimulation as well possible products from NO oxidation of dopamine and norepinephrine. Finally, a device that will allow the integration of the cell reactor with the analysis system will be developed using a on-chip valving network. While the majority of the work described in this proposal pertains to the separate parts of the integrated cell reactor/analysis system, the final device will result in a system that can be used in future work to study in detail the mechanisms leading to dopaminergic degeneration by NO.

描述(申请人提供)：这项提案的总体目标是开发一种基于微芯片的细胞培养模型，该模型与基于分离的分析系统相结合，最终研究一氧化氮(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一个特征，研究表明，小胶质细胞的炎症导致诱导型一氧化氮合酶(INOS)的激活，iNOS产生的NO可能进入多巴胺能细胞(如PC 12细胞)并氧化储存囊泡中的多巴胺。这种NO介导的氧化可能导致多巴胺储存枯竭，从而减少胞吐多巴胺的数量，这是帕金森患者的一个已知特征。这些发现可能导致NO介导的多巴胺能细胞损伤的可能机制；然而，对导致多巴胺能退化的机制缺乏分子水平的了解。由于在体内很难在分子水平上了解这些机制，因此通常采用细胞培养模型。为了深入了解这些机制，我们建议开发一个基于微芯片的系统，结合PC 12细胞来研究NO对多巴胺能变性的影响。为了实现这一点，一个三维的PC12细胞培养模型将被固定在一个平面微芯片上。通过监测钙刺激时释放的神经递质(多巴胺和去甲肾上腺素)，这些细胞将显示出生物反应。为了能够定量鉴定PC 12细胞释放的神经递质，将开发一种基于微芯片毛细管电泳法的分离系统。该系统将利用安培检测来监测细胞在刺激时释放的多巴胺和去甲肾上腺素，以及可能的多巴胺和去甲肾上腺素氧化产物。最后，将使用芯片上的阀门网络开发一种允许电池反应堆与分析系统集成的设备。虽然本提案中描述的大部分工作涉及集成电池反应堆/分析系统的单独部分，但最终的设备将产生一个系统，该系统可用于未来的工作，详细研究由NO导致多巴胺能退化的机制。