Laser Capture Microdissection System to Study Neuromuscular Biology

用于研究神经肌肉生物学的激光捕获显微切割系统

• 批准号: RTI-2019-00798
• 负责人: Ljubicic, Vladimir
• 金额: $ 10.93万
• 依托单位: McMaster University
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=657872
• 关键词: Laser; Capture; Microdissection; System; Study

Gene and protein expression profiling and the delineation of specific signaling pathways activated in response to a variety of stimuli within skeletal muscle have for the most part focused on the analysis of whole tissue extracts obtained from human biopsies or animal models. While these studies have yielded important insights into the aggregate response of skeletal muscle to various physiological stimuli, such approaches do not allow elucidation of the specific molecular responses of individual myofiber types, satellite cells, or capillaries within the muscle. Similarly, in animal models of neuromuscular adaptation, gene expression in motoneurons specifically is convoluted when whole spinal cords are analyzed. To address these knowledge gaps, we require a methodology to study cell-specific gene expression in muscles and spinal cord during conditions of phenotypic plasticity. Therefore, the purpose of this proposal is to obtain a laser capture microdissection (LCM) system. LCM is an approach that permits the rapid and reliable dissection and capture of homogeneous cell populations (e.g., myofibers, endothelium, motoneurons) or defined subcellular regions (e.g., neuromuscular junctions, myonuclear domains) without contamination from surrounding tissues, which enables a truly mechanistic approach for assaying the quantitative transcriptomic and proteomic status of these components in various phenotypes, as well as during transitions between these states. Few studies have investigated the cell-specific biology or subcellular biology of skeletal muscle or spinal cord in vivo. Furthermore, none have focused on mechanisms of gene expression during conditions of neuromuscular remodelling in basic rodent experiments, as well as in applied and basic human studies with particular emphasis on the effects of acute and chronic exercise in young and older males and females. Thus, acquisition of a LCM system represents a timely opportunity to innovate, to substantially enhance the research capabilities of our laboratories, as well as to expand HQP training opportunities using state-of-the-art technologies.

基因和蛋白质表达谱分析以及骨骼肌内响应于各种刺激而激活的特异性信号传导途径的描绘大部分集中在从人类活检或动物模型获得的全组织提取物的分析上。虽然这些研究已经对骨骼肌对各种生理刺激的聚集反应产生了重要的见解，但这些方法不允许阐明肌肉内单个肌纤维类型、卫星细胞或毛细血管的特定分子反应。类似地，在神经肌肉适应的动物模型中，当分析整个脊髓时，运动神经元中的基因表达特别是卷曲的。为了解决这些知识差距，我们需要一种方法来研究表型可塑性条件下肌肉和脊髓中的细胞特异性基因表达。因此，本提案的目的是获得激光捕获显微切割（LCM）系统。LCM是一种允许快速和可靠地解剖和捕获同质细胞群（例如，肌纤维、内皮、运动神经元）或确定的亚细胞区域（例如，神经肌肉接头，肌纤维结构域），而不受周围组织的污染，这使得能够实现用于测定这些组分在各种表型中以及在这些状态之间的转换期间的定量转录组学和蛋白质组学状态的真正的机械方法。很少有研究调查的细胞特异性生物学或亚细胞生物学的骨骼肌或脊髓在体内。此外，没有一个集中在基础啮齿动物实验中的神经肌肉重塑条件下的基因表达机制，以及在应用和基础人类研究中，特别强调急性和慢性运动对年轻和老年男性和女性的影响。因此，收购LCM系统是一个及时的创新机会，可以大大提高我们实验室的研究能力，并利用最先进的技术扩大HQP培训机会。