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，但针对的是单细胞，通常是悬浮的。该项目解决了在不牺牲单个细胞的情况下对它们进行分析化学的需求。由于单细胞电穿孔在细胞膜上创建瞬时端口，因此它是获取细胞质内容物样本的绝佳方法。脱离其上下文的单元格，例如自然贴壁细胞的悬浮液可能无法代表其自然状态，因此该项目重点关注贴壁细胞和组织，而不是悬浮细胞。我们最近发现培养物中的贴壁细胞非常坚固。即使在细胞质中失去大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，以便细胞质中所需的低分子量溶质部分（例如 20%）通过瞬时孔扩散。这一观察结果为在不杀死单细胞的情况下获取样本奠定了基础。在这个项目中，我们将开发用于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，并同时对灌注的细胞进行电穿孔。然后我们可以详细了解溶质进入或离开单细胞的传质速率。将开发另一种方法来测量培养的海马组织中的单细胞。它将应用于与中风和类似事件相关的重要问题，在这些事件中，流向大脑某个区域的血流暂时丧失。我们将建立这种方法来确定海马培养物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内容物。我们还将开发一种方法，基于对产生间隙连接的蛋白质进行 siRNA 的聚焦电穿孔，来削弱星形胶质细胞通过间隙连接相互通信的能力。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缺氧/葡萄糖剥夺后维持神经元谷胱甘肽水平很重要。 公共健康相关性：为了了解对损伤（尤其是缺血）的生化反应，需要用于控制和测量单细胞胞内和胞外空间化学成分的新工具。我们测量单细胞谷胱甘肽状态的方法不仅对研究缺血/再灌注具有深远的影响，而且对许多广泛存在的疾病，即阿尔茨海默病和帕金森病、精神分裂症和癫痫症也具有深远的影响。对组织培养物中的单细胞进行测量将有助于阐明星形胶质细胞在缺血/再灌注中对神经元健康的作用。