Single-cell sampling and analysis

了解细胞功能背后的分子机制是科学的主要目标。不幸的是，从血液或各种组织中获得的自然细胞群往往很难研究，因为它们只能在不同的混合物中获得。例如，在成人组织中发现的干细胞在医学上具有巨大的潜力，可以取代受损或患病的组织。然而，它们是稀有的，不能收获或培养到常规批量生化分析所需的大量数量。因此，单细胞分析对于研究和理解细胞行为以及基于干细胞的技术和疗法的发展至关重要。毛细管电泳(CE)在熔融硅柱或微芯片通道中进行，是一种能够基于小体积的大小和电荷对分析物进行高分辨率分离的分离技术(&lt；10^-9 L)。正因为如此，毛细管电泳法与激光诱导荧光检测方法的检测灵敏度提高相结合，是分析单个哺乳动物细胞的一种强有力的方法。这项提案的总体目标是开发和优化毛细管电泳法和微芯片电泳法进行单细胞生化分析的策略。第一个具体目标是通过优化细胞采样方法和分离柱的制造，最大限度地从特定的细胞室中回收分析物。第二个目标是证明单细胞毛细管电泳法分析在干细胞命运决定中起重要作用的膜系转录因子Notch的翻译后修饰。毛细管或微芯片上的单细胞电泳法正在成为生物发现工具的下一次革命。改进的采样和估计损失的能力将允许对细胞中的荧光分析物进行更灵敏和精确的定量。此外，一旦优化，单细胞CE将提供一个强大的手段来研究具有复杂翻译后调节的蛋白质，如Notch蛋白。因此，单细胞CE有可能成为针对Notch和干细胞的药物、基因或细胞疗法开发的重要工具。

Understanding the molecular mechanism underlying cellular function is a major goal in science. Unfortunately natural cell populations obtained from the blood or from various tissues are often difficult to study, as they can only be obtained in heterogeneous mixtures. For instance, stem cells found in adult tissues have a great potential in medicine for replacing damaged or diseased tissues. However, they are rare and cannot be harvested or grown to the large numbers needed for conventional bulk biochemical assays. Thus, single cell assays are essential for studying and understanding cell behaviour and the development of stem cell-based technologies and therapies. Capillary electrophoresis (CE), performed in fused-silica columns or on microchip channels, is a separation technology that is capable of high resolution separations of analytes based on size and charge from small volumes (< 10^-9 L). As such CE combined with the enhanced detection sensitivity of a laser-induced fluorescence detection scheme represents a powerful approach for the analysis of single mammalian cells. The overall objective of this proposal is to develop and optimize strategies for the biochemical analysis of single-cell by capillary and microchip electrophoresis. The first specific objective is to maximize the recovery of analytes from specific cell compartments by optimizing the method of cell sampling and the fabrication of the separation column. The second objective is to demonstrate the utility of single-cell CE to analyse posttranslational modifications of a membrane-tethered transcription factor important in stem cell fate decision, Notch. Single-cell electrophoresis in capillaries or on microchips is emerging as the next revolution in tools for biological discovery. Improved sampling and the ability to estimate losses will allow more sensitive and precise quantitation of fluorescent analytes in cells. Furthermore, once optimized, single-cell CE will provide a powerful means to study proteins that have complex posttranslational regulation such as the Notch protein. Consequently, single-cell CE has the potential to become an essential tool in development of drug, gene or cellular therapies targeting Notch and stem cells in general.