Laser Capture Microdissection System to Study Neuromuscular Biology

基因和蛋白质表达谱以及对骨骼肌内各种刺激激活的特定信号通路的描述，大部分集中在对从人体活组织检查或动物模型中获得的整个组织提取物的分析上。虽然这些研究对骨骼肌对各种生理刺激的总体反应产生了重要的见解，但这些方法并不能阐明肌肉中单个肌纤维类型、卫星细胞或毛细血管的特定分子反应。同样，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也会被扭曲。为了解决这些知识空白，我们需要一种方法来研究在表型可塑性条件下肌肉和脊髓中细胞特异性基因表达。因此，本提案的目的是获得一个激光捕获显微解剖（LCM）系统。LCM是一种方法，允许快速可靠地解剖和捕获均匀细胞群（例如，肌纤维，内皮，运动神经元）或定义的亚细胞区域（例如，神经肌肉连接，肌核结构域），而不受周围组织的污染，这使得一种真正的机制方法能够用于分析各种表型中这些成分的定量转录组学和蛋白质组学状态，以及这些状态之间的转换。很少有研究在体内对骨骼肌或脊髓的细胞特异性生物学或亚细胞生物学进行研究。此外，在基本的啮齿动物实验中，没有人关注神经肌肉重塑条件下基因表达的机制，以及在应用和基础的人类研究中，特别强调在年轻和老年男性和女性中急性和慢性运动的影响。因此，LCM系统的收购代表了一个及时的创新机会，大大提高了我们实验室的研究能力，并利用最先进的技术扩大了HQP培训的机会。

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.