Peckham Haley M, Ferner Anita H, Giuffrida Lauren, Murray Simon S, Xiao Junhua
Department of Anatomy and Neuroscience, The University of Melbourne.
Department of Anatomy and Neuroscience, The University of Melbourne; The Florey Institute of Neuroscience and Mental Health Research, The University of Melbourne.
J Vis Exp. 2015 Jan 12(95):52179. doi: 10.3791/52179.
Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). We use an in vitro myelination assay, an established model for studying CNS myelination in vitro. To do this, oligodendrocyte precursor cells (OPCs) are added to the purified primary rodent dorsal root ganglion (DRG) neurons to form myelinating co-cultures. In order to specifically interrogate the roles that particular proteins expressed by oligodendrocytes exert upon myelination we have developed protocols that selectively transduce OPCs using the lentivirus overexpressing wild type, constitutively active or dominant negative proteins before being seeded onto the DRG neurons. This allows us to specifically interrogate the roles of these oligodendroglial proteins in regulating myelination. The protocols can also be applied in the study of other cell types, thus providing an approach that allows selective manipulation of proteins expressed by a desired cell type, such as oligodendrocytes for the targeted study of signaling and compensation mechanisms. In conclusion, combining the in vitro myelination assay with lentiviral infected OPCs provides a strategic tool for the analysis of molecular mechanisms involved in myelination.
髓鞘形成是一个复杂的过程，涉及神经元和形成髓鞘的胶质细胞，即中枢神经系统（CNS）中的少突胶质细胞和周围神经系统（PNS）中的施万细胞。我们使用一种体外髓鞘形成检测方法，这是一种已确立的体外研究CNS髓鞘形成的模型。为此，将少突胶质前体细胞（OPC）添加到纯化的原代啮齿动物背根神经节（DRG）神经元中，以形成髓鞘化共培养物。为了具体探究少突胶质细胞表达的特定蛋白质在髓鞘形成中所起的作用，我们开发了一些方案，在将OPC接种到DRG神经元上之前，使用过表达野生型、组成型激活型或显性负性蛋白质的慢病毒选择性转导OPC。这使我们能够具体探究这些少突胶质细胞蛋白在调节髓鞘形成中的作用。这些方案也可应用于其他细胞类型的研究，从而提供一种方法，允许对所需细胞类型（如少突胶质细胞）表达的蛋白质进行选择性操作，用于信号传导和补偿机制的靶向研究。总之，将体外髓鞘形成检测方法与慢病毒感染的OPC相结合，为分析髓鞘形成所涉及的分子机制提供了一种策略性工具。
