Single Cell Electroporation

单细胞电穿孔

• 批准号: 8185059
• 负责人: STEPHEN G. WEBER
• 金额: $ 33.85万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8185059
• 关键词: Address; Alzheimer' s Disease; Analytical Chemistry; Astrocytes; Biochemical; Blood flow; Brain region; Cell Communication; Cell membrane; Cells; Chemicals; Communication; Complex; Connexin 43; Controlled Environment; Cytoplasm; Devices; Diffuse; Digitonin; Electrodes; Electroporation; Epilepsy; Extracellular Space; Foundations; Gap Junctions; Glucose; Glutathione; Glutathione Disulfide; Health; Hippocampus (Brain); Investigation; Ischemia; Label; Lead; Learning; Left; Life; MK-571; Maintenance; Measurement; Measures; Methods; Microfluidic Microchips; Microfluidics; Molecular Weight; Neuroglia; Neurons; Oxidation-Reduction; Oxygen; P-Glycoprotein; Parkinson Disease; Perfusion; Plasmids; Proteins; Reduced Glutathione; Reperfusion Therapy; Resolution; Role; Sampling; Schizophrenia; Small Interfering RNA; Stroke; Suspension substance; Suspensions; System; Techniques; Testing; Tissues; Transfection; base; cell killing; deprivation; improved; in vivo; inhibitor/antagonist; killings; knock-down; research study; response to injury; solute; tissue culture; tool

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.

描述（由申请人提供）：电穿孔是一种在细胞膜中产生瞬态孔的技术。它主要用于转染，并应用于细胞的悬浮液。单细胞电穿孔也用于转染，但通常用于悬浮液。该项目解决了在不牺牲单细胞上进行分析化学的必要性。由于单细胞电穿孔在细胞膜中产生瞬态端口，因此它是获得细胞质含量样品的绝佳方法。细胞从其上下文中取出，例如自然粘附细胞的悬浮液可能无法代表其自然状态，因此该项目着重于粘附的细胞和组织，而不是悬浮的细胞。我们最近发现，培养中的粘附细胞非常健壮。细胞在损失了细胞质中的低分子量溶质的大部分后仍能生存。我们还发现，我们可以控制单细胞电穿孔条件，从而使细胞质中低分子量溶质的所需部分（例如20％）通过瞬态孔扩散。该观察结果为从单个细胞中获取样品而无需杀死它们提供了基础。在这个项目中，我们将开发重要的单细胞生化研究工具。一种工具将能够用高空间分辨率灌注单粘细胞，并同时对被灌注的细胞进行电腐蚀。然后，我们可以详细了解进入或留下单个细胞的溶质的质量传输速率。将开发另一种方法来对培养的海马组织中的单个细胞进行测量。它将应用于与中风和类似事件有关的重要问题，在该问题中，流向大脑区域的血流暂时丢失。我们将建立这种方法，以确定海马培养中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含量。我们还将开发一种手段，以降低星形胶质细胞通过基于siRNA的局灶性电穿孔的间隙连接的能力，以创建间隙连接的蛋白质。我们将检验以下假设：相邻星形胶质细胞之间的溶质转运对于在氧气/葡萄糖剥夺后维持神经元谷胱甘肽水平很重要。 公共卫生相关性：需要单个细胞的细胞内和细胞外空间的化学组成的新工具才能理解生化对损伤的生化反应，尤其是缺血。我们对单个细胞的谷胱甘肽状态进行测量的方法不仅在研究缺血/再灌注方面具有深远的影响，而且在许多广泛的疾病中，即阿尔茨海默氏症和帕金森氏症的疾病，精神分裂症和癫痫病。对组织培养物中的单个细胞进行测量将导致阐明星形胶质细胞在缺血/再灌注中的神经元健康中的作用。