NSCs在OECs诱导下定向分化及分化后神经元电生理特性的研究

发布时间：2018-12-17 09:00

【摘要】： 目的：探索大鼠嗅鞘细胞(olfactory ensheathing cells, OECs)对神经干细胞(neural stem cells, NSCs)分化的影响,并记录分化后神经元电生理特性。 方法：①健康新生SD大鼠10只,取大脑皮层组织,用含2%B27、1%N2、20 ng／ml碱性成纤维生长因子(basic fibroblast growth factor, bFGF)和20 ng／ml表皮生长因子(epidermal growth factor, EGF)的培养液对NSCs进行悬浮培养,每5-7天传代一次,纯化培养2-3代备用,免疫细胞化学法对NSCs进行巢蛋白(Nestin)鉴定；②取新生SD大鼠嗅球,0.25%的胰蛋白酶和0.03%胶原酶37℃消化,改良Nash差速贴壁培养,加阿糖胞苷抑制杂细胞生长,当细胞数量达到大约80%时吸出上清,加入新鲜培养基(无血清DMEM),反复冲洗3遍,收集条件培养液,每隔3天半量换液。-20℃保存条件培养液,免疫细胞化学法对OECs进行神经生长因子受体p75(nerve growth factor receptor p75, NGFR p75)鉴定,直接免疫荧光流式细胞术检测嗅鞘细胞纯度；③实验组NSCs加OECs条件培养液及无血清DMEM／F12液培养,对照组NSCs只加无血清DMEM/F12培养液培养,每隔3天换液。观察细胞生长及分化情况,神经丝蛋白200(neurofilament200, NF200)鉴定神经元,胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)鉴定胶质细胞,膜片钳检测神经元电生理特性。 结果：新生SD大鼠大脑皮层中分离培养出NSCs,这些细胞具有长期增殖能力,免疫细胞化学鉴定显示Nestin染色阳性；新生SD大鼠嗅球培养出OECs,7天NGFR p75染色见大量阳性细胞,直接免疫荧光流式细胞术检测NGFR p75阳性细胞数目分别为94%、89%、97%(同一条件下检测)；对照组NSCs克隆球少部分贴壁,未见增殖分化,细胞逐渐萎缩,12天时细胞死亡,实验组2天后NSCs克隆球开始贴壁分化,7天时分化较明显,10天时免疫细胞化学染色,NF200染色显示NSCs克隆球大部分分化为神经元,GFAP染色显示NSCs克隆球少部分分化为胶质细胞；全细胞膜片钳检测显示分化后的神经元记录到快速激活快速失活、能被河豚毒素(tetrodotoxin, TTX)特异阻断的钠电流及慢激活慢失活、能被四乙基氯化铵(tetraethylammonium chloride, TEA)特异阻断的延迟整流性钾电流。 结论：①OECs能诱导NSCs向神经元方向分化；②分化后的神经元具有活跃的电生理特性,有替代凋亡、坏死神经元的潜能。
[Abstract]:Objective: To explore the effects of olfactive cells (OECs) on the differentiation of neural stem cells (NSCs) and to record the electrophysiological properties of the neurons after differentiation. Methods: 10 healthy newborn SD rats were cultured for 5-7 days with culture medium containing 2% B27, 1% N2, 20 ng/ ml of basic fibroblast growth factor (bFGF) and 20 ng/ ml of epidermal growth factor (EGF). 2-3 generations of NSCs were purified and cultured for 2-3 generations, and the NSCs were identified by immunocytochemical method. The olfactory bulb of neonatal SD rats, 0.25% of trypsin and 0.03% of collagenase were digested at 37 鈩，

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