基于雪旺细胞-背根节神经元共培养建立周围神经髓鞘化体外模型

An in vitro myelination model for peripheral nervous system developed in the Schwann cell-dorsal root ganglion neuron cocultures

目的:为探讨雪旺细胞与背根节(DRG)神经元髓鞘化共培养的标准化方法,并建立周围神经髓鞘化体外模型。方法:采用新生大鼠DRG神经节组织块培养法和胚胎大鼠DRG神经节分散培养法分别进行雪旺细胞和DRG神经元的培养和纯化,将雪旺细胞接种到培养DRG神经元的盖玻片上,按髓鞘化共培养程序进行雪旺细胞-DRG神经元共培养。结果:(1)雪旺细胞和DRG神经元的纯度、数量及比例;(2)共培养载体的包被基质;(3)髓鞘化共培养步骤和培养基成分是影响髓鞘形成的关键因素。原代雪旺细胞和DRG神经元S-100、神经元特异性烯醇化酶免疫荧光染色结果显示其纯度分别达到95%、90%;在髓鞘化培养基中共培养14d后相差显微镜和扫描电子显微镜下均观察到雪旺细胞包裹DRG神经元轴突形成髓鞘节段,髓鞘碱性蛋白免疫荧光染色结果显示免疫反应性;共培养体系和髓鞘结构可保持两个半月左右。结论:基于雪旺细胞和DRG神经元的条件化共培养能建立一种可靠的周围神经髓鞘化体外模型,为周围神经髓鞘化的主要调节因素及其作用机制研究提供有效的实验模型。

Objective：To develop a standardized method for myelinating cocultures of Schwann cell and dorsal root ganglion（DRG）neuron and establish an in vitro myelination model for peripheral nervous system.Methods：The primary rat Schwann cells were prepared by explanting dorsal root ganglions removed from neonatal rats,meanwhile the dissociated neuronal cultures consisted of rat embryonic day 15 DRG neurons were conducted.Schwann cell-DRG neuron cocultures were generated by seeding each coverslip of neurons with purified Schwann cells according to the myelinating coculture protocol.Results：Myelination in Schwann cell-DRG neuron coculture was influenced mainly by three factors,which were（1）purity,amount and proportion of Schwann cell and DRG neuron in cocultures,（2）matrix coated on the carrier of cocultures,and（3）procedure and medium composition used in myelinating coculture.Both the immunofluorescent staining of Schwann cell cultures using anti-S-100 antibodies,and that of DRG neuron cultures using anti-neuron specific enolase（NSE）antibodies demonstrated a high purity above 95% of primary Schwann cells,as well as 90% of DRG neurons.Schwann cells enwrapping the axon of DRG neurons and myelin segments were observed in cocultures after 14 days in myelinating medium by using phase contrast microscope and scanning electron microscope（SEM）,and myelin segments exhibited immunoreactivity when stained with anti-myelin basic protein（MBP）antibodies.The Schwann cell-DRG neuron cocultures and myelin structure could maintain for about two and a half months.Conclusion：The present results indicate that an in vitro myelination model which contributes to the investigation of the main factors involved in myelination in the peripheral nervous system has been established by means of conditioned cocultures of Schwann cells and DRG neurons.