【摘要】： 目的：探索大鼠嗅鞘細(xì)胞(olfactory ensheathing cells, OECs)對神經(jīng)干細(xì)胞(neural stem cells, NSCs)分化的影響,并記錄分化后神經(jīng)元電生理特性。 方法：①健康新生SD大鼠10只,取大腦皮層組織,用含2%B27、1%N2、20 ng／ml堿性成纖維生長因子(basic fibroblast growth factor, bFGF)和20 ng／ml表皮生長因子(epidermal growth factor, EGF)的培養(yǎng)液對NSCs進(jìn)行懸浮培養(yǎng),每5-7天傳代一次,純化培養(yǎng)2-3代備用,免疫細(xì)胞化學(xué)法對NSCs進(jìn)行巢蛋白(Nestin)鑒定；②取新生SD大鼠嗅球,0.25%的胰蛋白酶和0.03%膠原酶37℃消化,改良Nash差速貼壁培養(yǎng),加阿糖胞苷抑制雜細(xì)胞生長,當(dāng)細(xì)胞數(shù)量達(dá)到大約80%時(shí)吸出上清,加入新鮮培養(yǎng)基(無血清DMEM),反復(fù)沖洗3遍,收集條件培養(yǎng)液,每隔3天半量換液。-20℃保存條件培養(yǎng)液,免疫細(xì)胞化學(xué)法對OECs進(jìn)行神經(jīng)生長因子受體p75(nerve growth factor receptor p75, NGFR p75)鑒定,直接免疫熒光流式細(xì)胞術(shù)檢測嗅鞘細(xì)胞純度；③實(shí)驗(yàn)組NSCs加OECs條件培養(yǎng)液及無血清DMEM／F12液培養(yǎng),對照組NSCs只加無血清DMEM/F12培養(yǎng)液培養(yǎng),每隔3天換液。觀察細(xì)胞生長及分化情況,神經(jīng)絲蛋白200(neurofilament200, NF200)鑒定神經(jīng)元,膠質(zhì)纖維酸性蛋白(glial fibrillary acidic protein, GFAP)鑒定膠質(zhì)細(xì)胞,膜片鉗檢測神經(jīng)元電生理特性。 結(jié)果：新生SD大鼠大腦皮層中分離培養(yǎng)出NSCs,這些細(xì)胞具有長期增殖能力,免疫細(xì)胞化學(xué)鑒定顯示Nestin染色陽性；新生SD大鼠嗅球培養(yǎng)出OECs,7天NGFR p75染色見大量陽性細(xì)胞,直接免疫熒光流式細(xì)胞術(shù)檢測NGFR p75陽性細(xì)胞數(shù)目分別為94%、89%、97%(同一條件下檢測)；對照組NSCs克隆球少部分貼壁,未見增殖分化,細(xì)胞逐漸萎縮,12天時(shí)細(xì)胞死亡,實(shí)驗(yàn)組2天后NSCs克隆球開始貼壁分化,7天時(shí)分化較明顯,10天時(shí)免疫細(xì)胞化學(xué)染色,NF200染色顯示NSCs克隆球大部分分化為神經(jīng)元,GFAP染色顯示NSCs克隆球少部分分化為膠質(zhì)細(xì)胞；全細(xì)胞膜片鉗檢測顯示分化后的神經(jīng)元記錄到快速激活快速失活、能被河豚毒素(tetrodotoxin, TTX)特異阻斷的鈉電流及慢激活慢失活、能被四乙基氯化銨(tetraethylammonium chloride, TEA)特異阻斷的延遲整流性鉀電流。 結(jié)論：①OECs能誘導(dǎo)NSCs向神經(jīng)元方向分化；②分化后的神經(jīng)元具有活躍的電生理特性,有替代凋亡、壞死神經(jīng)元的潛能。
[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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