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上产生瞬时孔的技术。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，但是是在单细胞上，通常是悬浮的。这个项目解决了在不牺牲单个细胞的情况下对单个细胞进行分析化学的需要。由于单细胞电穿孔在细胞膜上产生瞬时端口，这是获得细胞质内容物样品的极好方法。脱离其环境的细胞，例如自然贴壁细胞的悬液可能不能代表其自然状态，因此该项目侧重于贴壁细胞和组织，而不是悬浮细胞。我们最近发现，贴壁细胞在培养中非常健壮。即使在细胞质中失去相当一部分低分子量溶质后，细胞仍能存活。我们还发现，我们可以控制单细胞电穿孔条件，使细胞质中所需的低分子量溶质比例（例如20%）通过瞬态孔扩散。这一观察结果为在不杀死单个细胞的情况下获得样本提供了基础。在这个项目中，我们将开发单细胞生化研究的重要工具。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细胞。这样我们就可以详细地了解溶质进入或离开单个细胞的质量传递速率。另一种方法将用于在培养的海马组织中对单个细胞进行测量。它将被应用于与中风和类似事件相关的一个重要问题，在这些事件中，流向大脑某个区域的血液暂时丢失。我们将建立这种方法来确定重要的谷胱甘肽氧化还原系统在海马培养的单个神经元中的地位。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通过间隙连接相互通信的能力，该方法基于siRNA的局部电穿孔，用于产生间隙连接的蛋白质。我们将验证相邻星形胶质细胞之间的溶质运输对于维持缺氧/葡萄糖剥夺后神经元谷胱甘肽水平的重要假设。