Microchip-based Cell Reactor/Analysis System

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系统，该系统集成了细胞培养模型和基于分离的分析系统，以研究一氧化氮(NO)对多巴胺能变性的影响。多巴胺能神经元的退化是帕金森氏病的一个特征，研究表明，小胶质细胞的炎症导致诱导型一氧化氮合酶的激活，该酶产生的NO可能进入多巴胺能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。鉴于细胞内神经元变化可能与帕金森病的发生有关的所有已知情况，对于NO导致多巴胺能变性的机制在分子水平上仍缺乏详细的了解。研究这些机制的最佳情况是使用体外细胞培养系统，该系统模仿体内系统，并易于与分析系统相结合，该分析系统可以离散地分析当没有耗尽多巴胺储存时形成的产品。在这个方案中，我们描述了使用微芯片技术和PC12单元来开发这样的系统。目前获得资金的赠款在发展这一系统方面取得了很大进展。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，该系统结合了PC 12细胞的固定、芯片上的瓣膜、基于电泳的分离和电化学检测。为了实现这一目标，我们首先将PC-12细胞固定化和安培检测与本研究开发的连续流动反应器/芯片电泳仪相结合。固定化条件和芯片尺寸等变量将被优化，使细胞在圆形微通道中接近融合。然后，我们建议使用这个装置，通过监测细胞与不同浓度的NO孵育时多巴胺和去甲肾上腺素释放的变化，来研究NO对多巴胺能退化的影响。我们还建议确定NO与这些神经递质相互作用的不同产物。最后，我们建议通过在微芯片设备中同时培养PC 12细胞和小胶质细胞来开发更现实的帕金森发病模型，长期目标是在这些研究中使用小胶质细胞作为NO来源。这将包括优化小胶质细胞的固定条件，以及表明小胶质细胞具有生物响应性的研究。虽然这项提议的重点是PC 12细胞和神经递质分析，但最终的设备将是一种通用的分析工具，可以固定各种细胞系，并通过电泳手段分析释放的分析物。 这项提议的总体目标是开发一种微芯片设备，可以用来研究一氧化氮在多巴胺能变性中所起的作用，多巴胺能变性是帕金森病的一个特征。这个最终的装置将利用微米大小的阀门将神经细胞模型集成到分析系统中，该系统可以区分细胞释放的神经递质。这一设备将使神经科学家能够研究小胶质细胞在帕金森氏症发病中所起的作用。

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.