嗅鞘细胞诱导神经干细胞分化后神经元电生理特性的研究

Electrophysiological characters of the neurons from differentiation of neural stem cells induced by olfactory ensheathing cells

目的探索大鼠嗅鞘细胞对神经干细胞(NSC)分化的影响,以及分化后神经元电生理特性。方法取新生鼠大脑皮质,原代培养大鼠NSC。NSC分为实验组和对照组,实验组将无血清培养的NSC中加入嗅鞘细胞条件培养液,对照组单纯无血清培养NSC。光镜下观察细胞分化情况,免疫组化法分别检测巢蛋白(nestin)、神经生长因子受体(NGFRp75)、神经丝蛋白(NF200)和胶质纤维酸性蛋白(GFAP)的表达,膜片钳检测神经元电生理特性。结果实验组嗅鞘细胞主要诱导NSC分化为神经元,少量分化为胶质细胞。对照组NSC逐渐萎缩,最终死亡。分化后的神经元记录到快速激活、快速失活能被河豚毒素特异阻断的钠电流,以及慢激活、慢失活能被四乙铵特异阻断的延迟整流性钾电流。结论嗅鞘细胞能诱导NSC分化成神经元,分化后的神经元具有活跃的电生理特性。

Objective To study the effect of olfactory ensheathing cells（OECs）on differentiation of neural stem cells（NSCs）and electrophysiological characters of neurons after differentiation.Methods Primary NSCs cultures were obtained from newborn rat cerebral cortex.The NSCs were divided into experimental group and control group.NSCs cultured in the serum-free medium to which conditioned medium of OECs was added to served as experimental group and NSCs in serum-free medium alone as control group.The differentiation of NSCs was observed by light microscopy.The expression of nestin,nerve growth factor receptor（NGFR p75）,neurofilament（NF200）and glial fibrillary acidic protein（GFAP）were analyzed by immunocytochemistry.The electrophysiological characters of neurons differentiated from NSCs were detected by patch clamp.Results In experimental group,NSCs were induced mainly into neurons and the minority of them differentiated into glial cells in the experimental group.The NSCs became atrophic gradually and finally died in control group.The rapidly activated and deactivated sodium current which could be blocked by specific blocker of sodion Tetrodotoxin was recorded,so did the slowly activated and deactivated delayed rectifier potassium current which could be blocked by specific blocker of potassium ion tetraethylamine.Conclusions OECs can induce NSCs into neurons which have active electrophysiological properties.