Schott Jason T, Kirby Leslie A, Calabresi Peter A, Baxi Emily G
Neurology, Johns Hopkins University, School of Medicine.
Neurology, Johns Hopkins University, School of Medicine;
J Vis Exp. 2016 Feb 17(108):53764. doi: 10.3791/53764.
Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as multiple sclerosis and transverse myelitis. Myelination is required to not only facilitate rapid impulse propagation within the central nervous system, but also to provide trophic support to underlying axons. Oligodendrocyte progenitor cells (OPCs) can be studied in vitro to help identify factors that may promote or inhibit oligodendrocyte differentiation. To date, many of the methods available to evaluate this process have either required large numbers of cells, thus limiting the number of conditions that can be investigated at any one time, or labor-intensive methods of quantification. Herein, we describe a protocol for the isolation of large numbers of highly pure OPCs together with a fast and reliable method to determine oligodendrogenesis from multiple conditions simultaneously. OPCs are isolated from P5-P7 neonatal rat cortices and grown in vitro for three days prior to differentiation. Four days after differentiation, oligodendrogenesis is evaluated using a dual-infrared fluorescence-scanning assay to determine expression of the myelin protein.
高效的少突胶质细胞生成是包括脊髓损伤、新生儿缺氧以及脱髓鞘疾病（如多发性硬化症和横贯性脊髓炎）等多个研究领域的治疗目标。髓鞘形成不仅需要促进中枢神经系统内的快速冲动传播，还需要为下层轴突提供营养支持。少突胶质前体细胞（OPCs）可以在体外进行研究，以帮助确定可能促进或抑制少突胶质细胞分化的因素。迄今为止，许多用于评估这一过程的方法要么需要大量细胞，从而限制了在任何给定时间可研究的条件数量，要么是劳动密集型的定量方法。在此，我们描述了一种分离大量高纯度OPCs的方案，以及一种快速可靠的方法，可同时从多种条件下确定少突胶质细胞生成情况。从出生后第5 - 7天的新生大鼠皮质中分离OPCs，并在体外培养三天后进行分化。分化四天后，使用双红外荧光扫描测定法评估少突胶质细胞生成，以确定髓鞘蛋白的表达。
