Department of Neurology, The Second Affiliated Hospital of Nanchang University, Nanchang, China.
Artif Cells Nanomed Biotechnol. 2020 Dec;48(1):298-304. doi: 10.1080/21691401.2019.1671428.
This study aimed to investigate the effect of Fyn gene silencing on the apoptosis of oligodendrocytes (OLs) in epileptic model and the involved mechanism. Primary oligodendrocyte pro-genitor cells (OPCs) were separated from rats and differentiated to OLs. Immunofluorescent labeling showed positive expression of A2B5 in OPCs and Olig2 in OLs, suggesting the successful separation of OPCs and OLs. Three Fyn siRNAs (si-Fyn) and Fyn siRNA negative control (NC) were transfected into OLs. Western blot showed that among three si-Fyn groups, si-Fyn3 caused the lowest Fyn expression, so si-Fyn3 was chosen for following experiment. Cells were divided into four groups: Control, Model, NC and si-Fyn. In the Model group, cells were cultured in Mg-free extracellular fluid for 3 h. The morphology of control cells was normal. However, the migration of neurons, the aggregation of cell bodies and the "grid-like" changes of neural networks were observed in the model cells. OLs apoptosis in various groups was assessed by flow cytometry. Expression of Fyn, ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2) in OLs of various groups was evaluated by western blot. Compared with the Control group, the apoptotic rates, the Fyn expression and p-ERK1/2/ERK1/2 ratio in the Model and NC groups increased significantly ( < .05). However, the apoptotic rate, the Fyn expression and p-ERK1/2/ERK1/2 ratio in the si-Fyn group were remarkably smaller than those in the Model group ( < .05). In conclusion, Fyn gene silencing reduced the apoptosis of OLs through inhibiting the phosphorylation of ERK1/2 in epileptic model.
本研究旨在探讨 Fyn 基因沉默对癫痫模型中少突胶质细胞（OLs）凋亡的影响及其机制。从大鼠中分离出原代少突胶质前体细胞（OPCs）并分化为 OLs。免疫荧光标记显示 OPCs 中 A2B5 阳性表达，OLs 中 Olig2 阳性表达，提示 OPCs 和 OLs 分离成功。将三种 Fyn siRNA（si-Fyn）和 Fyn siRNA 阴性对照（NC）转染至 OLs 中。Western blot 显示，在三个 si-Fyn 组中，si-Fyn3 引起的 Fyn 表达最低，因此选择 si-Fyn3 进行后续实验。细胞分为四组：对照组、模型组、NC 组和 si-Fyn 组。在模型组中，细胞在无镁细胞外液中培养 3 h。对照组细胞形态正常，而模型组细胞中神经元迁移、细胞体聚集和神经网络“网格状”改变明显。通过流式细胞术评估各组 OLs 的凋亡情况。Western blot 评估各组 OLs 中 Fyn、ERK1/2 和磷酸化 ERK1/2（p-ERK1/2）的表达。与对照组相比，模型组和 NC 组的凋亡率、Fyn 表达和 p-ERK1/2/ERK1/2 比值显著升高（ < .05）。然而，si-Fyn 组的凋亡率、Fyn 表达和 p-ERK1/2/ERK1/2 比值明显小于模型组（ < .05）。综上所述，Fyn 基因沉默通过抑制癫痫模型中 ERK1/2 的磷酸化减少 OLs 的凋亡。
