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Purification and Characterization of Mature Murine and Human Satellite Glial Cells

成熟鼠和人卫星胶质细胞的纯化和表征

基本信息

批准号：
9760015
负责人：
Temugin Berta
金额：
$ 20.06万
依托单位：
UNIVERSITY OF CINCINNATI
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9760015
关键词：
Ablation Acceleration Acute Adjuvant Study Adult Afferent Neurons American Animal Model Anti-inflammatory Antibodies Astrocytes Biochemical Bioinformatics Calcium Cell Adhesion Cells Cellular biology Cytokine Receptors Data Databases Development Disease Exhibits Exposure to Freund&apos s Adjuvant Functional disorder Future Gene Expression Profile Gene Expression Profiling Gene Silencing Genes Genetic Transcription Glycoproteins Goals Headache Health Homeostasis Human Image Immune Immunohistochemistry In Situ Hybridization In Vitro Individual Inflammation Inflammatory Interleukin-10 Interleukin-4 Investigation Lead Methods Microglia Molecular Molecular Profiling Mus Neuroglia Neurons Outcome Pain Pain Research Pain management Pathogenicity Pattern Pharmaceutical Preparations Pharmacology Population Prevalence Procedures Protocols documentation Public Health RNA Research Research Personnel Resolution Reverse Transcriptase Polymerase Chain Reaction Role Sensory Ganglia Specificity Spinal Ganglia Surface Suspensions TNF gene Temporomandibular Joint Disorders Testing Therapeutic Time Trigeminal System Universities Work base cell type chronic pain chronic painful condition collaborative environment cytokine genetic manipulation genetic signature human tissue in vitro Model inflammatory milieu inflammatory pain innovation insight interest mouse model new therapeutic target novel novel strategies novel therapeutic intervention novel therapeutics optogenetics pain processing painful neuropathy receptor reduce symptoms response screening side effect tool transcriptome sequencing translational approach

项目摘要

More than 100 million Americans suffer from chronic pain or severe pain. However, current pain treatments are inefficient and accompanied by undesirable side effects. Both inflammation and satellite glial cells (SGCs) in sensory ganglia are common features of chronic pain such as headache, temporomandibular joint disorders, neuropathic and inflammatory pain, and they may represent new therapeutic targets. However, there is a lack of adequate tools for the direct interrogation of SGCs from adult individuals and their interactions with inflammation. In this proposal we have established a new and transformative tool for the study of adult mouse and human SGCs by immunopanning, which consists in passing a cell suspension over several dishes coated with specific antibodies to deplete unwanted cell types, and a final antibody-coated dish being used to select the cell type of interest. Immunopanning is relatively simple, inexpensive, gentle on cells, and can produce very high yields of purified cells for transcriptional, biochemical and pharmacological analyses. Our long-term goal is to better understand how maladaptive perturbations between neurons, glial and immune cells can participate in pain and how they can be targeted for the development of more effective and safer therapeutic approaches. The main objective of this application is to establish a reliable protocol for the characterization and understanding of the inflammatory pathophysiology of adult murine and human SGCs. Our central hypothesis is that acutely isolated SGCs from adult mice and humans exhibit a unique molecular profile that can be modulated by inflammation to display either pathogenic or protective functions. This hypothesis will be tested by pursuing the following specific aims: (1) Identification of a unique transcriptional signature in murine and human SGCs; and (2) Characterization of murine and human SGC responses to inflammation. These aims will be accomplished by immunopanning and RNA sequencing in combination with bioinformatics, immunohistochemical, calcium imaging, in situ hybridization and real-time RT-PCR approaches. The proposed work is innovative because it will challenge conventional concepts that are biased toward exclusive neuronal mechanisms and therapies of pain and provide, for the first time, a comprehensive, unbiased analysis of the pattern of gene expression and functional characterization of mouse and human SGCs in health and disease conditions. The outcomes of these investigations will be (1) the establishment of a simple and relatively inexpensive procedure to isolate a large, and highly enriched population of mature SGCs, (2) the identification of the molecular repertoire of mouse and human SGCs, (3) the determination of the functions of inflammation and SGCs in a mouse model of chronic pain, and (4) the establishment of an in vitro model to study these functions in mouse and human SGCs. The application is particularly significant because these sets of tools and databases should greatly benefit the pain research field and accelerate the development of new therapeutics to efficiently suppress chronic pain.

超过1亿美国人患有慢性疼痛或严重疼痛。然而，目前的疼痛治疗是效率低，并伴有不希望的副作用。炎症和卫星神经胶质细胞（SGCs），感觉神经节是慢性疼痛如头痛，颞下颌关节紊乱，神经性和炎性疼痛，它们可能代表新的治疗靶点。然而，缺乏足够的工具，直接询问的SGCs从成年人和他们的相互作用与炎症。在这个提议中，我们建立了一个新的和变革性的工具，用于成年小鼠和人类的研究。 SGCs通过免疫淘选，其包括使细胞悬浮液通过几个用特异性抗体包被的培养皿。抗体以消耗不需要的细胞类型，并且最终的抗体包被的培养皿用于选择细胞类型，兴趣免疫淘选相对简单、便宜、对细胞温和，并且可以产生非常高的产量。用于转录、生物化学和药理学分析的纯化细胞。我们的长期目标是更好地了解神经元，神经胶质细胞和免疫细胞之间的适应不良扰动如何参与疼痛，如何将它们作为目标，以开发更有效和更安全的治疗方法。主要本申请的目的是建立一种可靠的协议，用于表征和理解成年鼠和人SGCs的炎性病理生理学。我们的核心假设是，来自成年小鼠和人类的SGCs表现出独特的分子特征，可以通过炎症显示致病或保护功能。这一假设将通过追踪（1）在鼠和人SGCs中鉴定独特的转录标记; 和（2）鼠和人SGC对炎症反应的表征。这些目标将会实现通过免疫淘选和RNA测序结合生物信息学、免疫组织化学、钙离子成像、原位杂交和实时RT-PCR方法。这项工作是创新的，因为它将挑战传统观念，这些观念偏向于疼痛的神经机制和治疗并首次对基因表达模式进行全面、公正的分析，小鼠和人SGCs在健康和疾病条件下的功能表征。这些成果调查将是（1）建立一个简单和相对便宜的程序，和高度富集的成熟SGCs群体，（2）鉴定小鼠的分子库，（3）慢性炎症小鼠模型中炎症和SGCs功能的测定疼痛，和（4）建立体外模型来研究小鼠和人SGCs中的这些功能。的应用程序尤其重要，因为这些工具和数据库集将极大地帮助解决研究领域，并加速新疗法的开发，以有效地抑制慢性疼痛。