Single Cell Electroporation

描述（由申请人提供）：电穿孔是一种在细胞膜中产生瞬时孔的技术。它主要用于转染，并应用于细胞悬浮液。单细胞电穿孔也用于转染，但在单细胞上，通常在悬浮液中。该项目解决了在不牺牲单细胞的情况下对单细胞进行分析化学的需求。由于单细胞电穿孔在细胞膜中产生瞬时端口，因此它是获得细胞质内容物样品的极好方法。从其背景中取出的细胞，例如天然粘附细胞的悬浮液可能不能代表其天然状态，因此该项目侧重于粘附细胞和组织，而不是悬浮细胞。我们最近发现培养中的贴壁细胞非常健壮。细胞甚至在细胞质中损失了显著部分的低分子量溶质后仍存活。我们还发现，我们可以控制单细胞电穿孔条件，使得细胞质中所需部分的低分子量溶质，例如，20%，通过瞬态孔隙扩散。这一观察结果为从单细胞中获得样品而不杀死它们提供了基础。在这个项目中，我们将开发单细胞生物化学研究的重要工具。一种工具将能够以高空间分辨率灌注单个贴壁细胞并同时电穿孔灌注的细胞。然后，我们可以详细了解溶质进入或离开单细胞的质量传输速率。另一种方法将被开发用于对培养的海马组织中的单细胞进行测量。它将被应用于一个重要的问题，涉及中风和类似的事件，其中血液流向大脑的一个区域是暂时失去。我们将建立这种方法来确定在海马培养的单个神经元中重要的谷胱甘肽氧化还原系统的状态。这包括通过电穿孔和基于微流体的衍生化、分离和定量获得细胞质内容物。我们还将开发一种方法，以减少星形胶质细胞的能力，通过间隙连接的基础上，对siRNA的蛋白质，创造差距连接的局部电穿孔相互沟通。我们将测试的假设，相邻的星形胶质细胞之间的溶质转运是重要的神经元谷胱甘肽水平的维持后氧/葡萄糖剥夺。 公共卫生相关性：需要新的工具来控制和测量单细胞的细胞内和细胞外空间的化学组成，以了解损伤，特别是缺血的生化反应。我们的方法，使单细胞的谷胱甘肽状态的测量具有深远的影响，不仅研究缺血/再灌注，而且在一些广泛的条件，即阿尔茨海默氏症和帕金森氏症，精神分裂症，癫痫。对组织培养物中的单细胞进行测量将澄清星形胶质细胞在缺血/再灌注中对神经元健康的作用。

DESCRIPTION (provided by applicant): Electroporation is a technique that creates transient pores in cell membranes. It is mostly used for transfection, and applied to suspensions of cells. Single-cell electroporation is also used for transfection but on single cells, typically in suspension. This project addresses the need to do analytical chemistry on single cells without sacrificing them. As single-cell electroporation creates transient ports in cell membranes, it is an excellent approach to obtaining samples of cytoplasmic contents. Cells taken out of their context, e.g. suspensions of naturally adherent cells may not be representative of their natural state, so the project focuses on adherent cells and tissues, not on suspended cells. We have recently found that adherent cells in culture are remarkably robust. Cells survive even after losing a significant fraction of the low-molecular weight solutes in the cytoplasm. We have also found that we can control single-cell electroporation conditions so that a desired fraction of the low-molecular weight solutes in the cytoplasm, e.g., 20%, diffuses through the transient pores. This observation provides the foundation for obtaining samples from single cells without killing them. In this project, we will develop significant tools for single-cell biochemical investigations. One tool will be able to perfuse single adherent cells with high spatial resolution and simultaneously electroporate the perfused cell. We can then learn in detail the mass transport rates for solutes entering or leaving single cells. Another method will be developed for making measurements on single cells in cultured hippocampal tissue. It will be applied to an important question related to stroke and similar incidents in which blood flow to a region of the brain is temporarily lost. We will establish this method for determining the status of the important glutathione redox system in a single neuron in a hippocampal culture. This includes obtaining cytoplasmic contents by electroporation and microfluidic-based derivatization, separation, and quantitation. We also will develop a means to diminish the astrocytes' ability to communicate with each other through gap junctions based on focal electroporation of siRNA for the protein that creates the gap junctions. We will test the hypothesis that solute transport between adjacent astrocytes is important for maintenance of neuronal glutathione levels following oxygen/glucose deprivation. PUBLIC HEALTH RELEVANCE: New tools for controlling and measuring the chemical composition of the intra- and extracellular space of single cells are required for understanding biochemical responses to injury, especially ischemia. Our approach to making measurements of the glutathione status of single cells has far-reaching implications not only for studying ischemia/reperfusion, but also in a number of widespread conditions, namely Alzheimer's and Parkinson's diseases, schizophrenia, and epilepsy. Making measurements on single cells in tissue cultures will lead to a clarification of the role of astrocytes on neuronal health in ischemia/reperfusion.