少突胶质细胞分化异常与癫痫发病机制的相关性研究

A lot of previous studies has shown the correlativity between oligodendrocytes (OL) dysplasia and epilepsy. In this study, amygdala kindling epilepsy model in rats were constructed, myelinogenesis and oligodendrocytes(OL) differentiation in different period in the model were observed by neuropathological method and immunofluorescence technology, then the proteins related to myelinogenesis and OL development such as Smad interacting protein 1(SIP1), Oligodendrocyte transcription factor(Olig) and Fyn transcription and expression were detected, and the epilepsy mechanism about abnormal OL differentiation was analyzed. In the experiment, Olig1 gene silencing letivirus, Olig2 gene silencing letivirus, SIP1 over-expression letivirus and Fyn inhibitor were used respectively for the intervention of OL cutlure in vitro, and it was suggested that SIP1 might regulate OL differentiation through Fyn-Olig1/2 signaling pathway. Co-culture of OL cultured in vitro and neuron of epileptiform discharges was carried out, then OL-secreted neuroprotective factors were detected and neuron injured degree was observed by use of letiviral over-expression SIP1, patch-clamping techniques, specific staining and morphological observation, and the effects of neuron of abnormal discharges on OL differentiation and the regulating roles of SIP1 were analyzed.

大量前期研究发现少突胶质细胞（OL）发育异常与癫痫的相关性，现计划以杏仁核点燃癫痫模型为研究对象，应用神经病理及免疫荧光方法观察髓鞘形成及OL在该模型中不同时期的分化状态，检测髓鞘相关蛋白及OL发育相关蛋白：Smad蛋白相互作用蛋白1（SIP1）、OL转录因子（Olig）、Fyn的转录及表达，分析OL发育异常在癫痫发病中的作用机制。用Olig1、Olig2基因沉默慢病毒、SIP1过表达慢病毒及Fyn抑制剂分别干预原代培养OL，分析SIP1可能通过Fyn-Olig1/2信号通路调控OL分化。将原代培养的OL与癫痫样放电的神经元共培养，借助慢病毒过表达SIP1、膜片钳、细胞形态学观察及特殊染色等方法，检测OL分泌的神经保护因子，观察神经元损伤程度，分析异常放电神经元对OL分化的影响及SIP1对该过程的调控作用。

癫痫是神经系统重要疾病之一，在中国有大约一千万患者，而在全球影响了50亿人的生活质量，研究发现脱髓鞘或髓鞘再生障碍与癫痫的发生密切相关，但其发病机制仍需探讨。本研究通过动物实验及细胞实验观察癫痫演变过程中少突胶质细胞（OL）及髓鞘变化，进一步探讨SIP1介导OL分化可能机制，观察该过程中作用蛋白SIP1、Fyn、Olig1、Olig2在癫痫发病中可能发挥的作用。动物实验发现癫痫模型鼠癫痫发作后，LFB染色观察到髓鞘脱失，电镜下有部分髓鞘分层、融合、断裂，OL发育障碍出现在造模后3小时，MBP、MAG mRNA转录水平及蛋白表达水平降低，Olig1、Olig2、SIP1、Fyn蛋白表达均降低，与假手术组及对照组相比差异有统计学意义（P＜0.05）。构建SIP1、Olig1、Olig2干扰慢病毒载体，细胞实验发现过表达SIP1、Olig1、Olig2可促进OPC向OL细胞分化；干扰Olig2基因可抑制SIP1过表达基因引起的促分化作用，抑制Fyn的表达可抑制OPC分化成OL。神经元过度放电会抑制OPC分化成OL，成熟的OL可增加neurotrophin、TGF-β、FGF-9的表达来修复因癫痫样放电引起的神经元损伤，OL分化异常影响其修复受损神经元的功能。过表达SIP1可促进OPC分化成OL，恢复癫痫样放电模型对OPC分化的抑制作用，增加neurotrophin、TGF-β、FGF-9的表达来修复因癫痫样放电引起的神经元损伤。研究结果提示癫痫发作与OL发育异常有关， OL成熟障碍导致神经营养因子分泌减少导致神经元损伤，SIP1通过Fyn-Olig1/2信号通路调控OL分化，过表达SIP1可促进OL分化成熟，修复神经元损伤。