【摘要】： 目的 建立體外純化培養(yǎng)嗅鞘細(xì)胞(Olfactory ensheathing cells, OECs)的穩(wěn)定方法并觀察培養(yǎng)的嗅鞘細(xì)胞形態(tài)學(xué)特征;比較大鼠嗅鞘細(xì)胞與星型膠質(zhì)細(xì)胞在體外對(duì)神經(jīng)干細(xì)胞(Neural stem cells ,NSCs)分化的影響。 方法 分別從新生SD大鼠的嗅球采用小劑量阿糖胞苷分次純化法分離、培養(yǎng)嗅鞘細(xì)胞;從海馬、大腦皮質(zhì)分離、培養(yǎng)星型膠質(zhì)細(xì)胞、神經(jīng)干細(xì)胞,觀察細(xì)胞的形態(tài)發(fā)育特點(diǎn)及免疫組化特性。收集嗅鞘細(xì)胞及其上清液、星型膠質(zhì)細(xì)胞及其上清液,將實(shí)驗(yàn)分為5組:組Ⅰ:OECs上清液+ NSCs;組Ⅱ: OECs+ NSCs;組Ⅲ:星型膠質(zhì)細(xì)胞上清液+ NSCs;組Ⅳ:星型膠質(zhì)細(xì)胞+ NSCs;組Ⅴ:NSCs單獨(dú)培養(yǎng)作為對(duì)照;將NSCs克隆球平均分成5份,加入各組,不加b-FGF、EFGF、B27,組Ⅰ和組Ⅲ分別用培養(yǎng)了OECs、星形膠質(zhì)細(xì)胞24h的DMEM/F12 ( 1:1 )上清液3ml培養(yǎng),每24h換液,組Ⅱ和組Ⅳ、組Ⅴ用DMEM/F12 ( 1:1 ) 3ml培養(yǎng),每24h換液,各組置于相同條件培養(yǎng)箱培養(yǎng),倒置顯微鏡下動(dòng)態(tài)觀察各組NSCs被誘導(dǎo)分化的細(xì)胞的生長(zhǎng)情況,并采用免疫組化法對(duì)分化細(xì)胞鑒定和計(jì)數(shù)。 結(jié)果 1、原代培養(yǎng)后24小時(shí)OECs形態(tài)基本可辨,背景可見成纖維細(xì)胞,第2天開始予小劑量阿糖胞苷分次純化后成纖維細(xì)胞大大減少,OECs的外形主要以突起細(xì)長(zhǎng)的雙極和三極為主,或無(wú)突起呈“煎蛋樣”,隨時(shí)間延長(zhǎng)細(xì)胞生長(zhǎng)排列呈現(xiàn)一定方向性;細(xì)胞免疫化學(xué)示大部分細(xì)胞神經(jīng)生長(zhǎng)因子受體P75(neurotrophin receptor p75,NGFRp75)陽(yáng)性表達(dá)。 2、比較大鼠嗅鞘細(xì)胞與星型膠質(zhì)細(xì)胞在體外對(duì)NSCs分化的影響結(jié)果發(fā)現(xiàn):組Ⅰ、組Ⅱ的NSCs球6h已開始貼壁生長(zhǎng),至第7天,多數(shù)分化為具有2-3個(gè)長(zhǎng)突起,胞體圓形或橢圓形的神經(jīng)元樣細(xì)胞, NF200 (1:150 )免疫組織化學(xué)結(jié)果顯示這類細(xì)胞胞體甚至突觸皆被染成棕黃色,胞核不染色,為強(qiáng)陽(yáng)性,證明該類細(xì)胞為神經(jīng)元,計(jì)算其神經(jīng)元分化率:組Ⅰ為( 51.43±8.58) %,組Ⅱ?yàn)?50.11±9.56) % ;組Ⅲ、組Ⅳ大部分細(xì)胞NF200 ( 1:150 )染色陰性,GFAP ( 1:150 )陽(yáng)性,其神經(jīng)元分化率分別為( 17.25±3.01 ) %、( 15.51±1.62 ) %;組Ⅴ的NSCs球大多數(shù)在3-4天后萎縮、漂浮,5-7天基本死亡,神經(jīng)元分化率為( 0.56±0.33 ) %。經(jīng)統(tǒng)計(jì)學(xué)分析嗅鞘細(xì)胞組的神經(jīng)元分化率遠(yuǎn)遠(yuǎn)高于星型膠質(zhì)細(xì)胞組及對(duì)照組的神經(jīng)元分化率(P 0.01 )。 3、結(jié)論 1、A ra-C抑制法穩(wěn)定易復(fù)制,可獲得高純度的OECs。 2、大鼠嗅鞘細(xì)胞(OECs)較星型膠質(zhì)細(xì)胞在體外能更好地促進(jìn)神經(jīng)干細(xì)胞(NSCs)分化為神經(jīng)元。
[Abstract]:Objective to establish a stable method for the purification and culture of olfactory ensheathing cells (Olfactory ensheathing cells, OECs) in vitro and to observe the morphological characteristics of cultured olfactory ensheathing cells. To compare the effects of olfactory ensheathing cells and astrocytes on (Neural stem cells, NSCs) differentiation of neural stem cells in vitro. Methods olfactory ensheathing cells were isolated from olfactory bulb of newborn SD rats by small dose cytarabine purification. Astrocytes and neural stem cells were cultured from hippocampus and cerebral cortex. The morphological and immunohistochemical characteristics of the cells were observed. Olfactory ensheathing cells and their supernatants, astrocytes and their supernatants were collected and divided into five groups: group 鈪，

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