本文選題：骨髓間充質(zhì)干細(xì)胞 + 褪黑素��； 參考：《江蘇大學(xué)》2009年碩士論文

【摘要】： 目的研究大鼠骨髓間充質(zhì)干細(xì)胞(Marrow Mesenchymal Stem Cells,MSCs)經(jīng)分離純化、體外培養(yǎng)后,以褪黑激素(Melatonin,MT)、嗅鞘細(xì)胞(OlfactoryEnsheathing Cells,OECs)上清液誘導(dǎo)MSCs向神經(jīng)元樣細(xì)胞分化的情況。探討MT體外誘導(dǎo)MSCs不同時段向神經(jīng)樣細(xì)胞分化的情況,以及MT、OECs上清液聯(lián)合誘導(dǎo)的優(yōu)劣,找出二者最優(yōu)誘導(dǎo)方案。為更好的研究MSCs向神經(jīng)元樣細(xì)胞分化提供一定的實驗基礎(chǔ),為細(xì)胞移植提供又一類種子細(xì)胞。 方法在無菌條件下,取健康雄性SD大鼠的骨髓組織,用密度為1.077g/mL的Ficoll人淋巴細(xì)胞分離液密度梯度離心法獲取有核細(xì)胞層,以5×10~6/cm~2密度接種于25ml細(xì)胞培養(yǎng)瓶,用含10%胎牛血清的L-DMEM培養(yǎng)基懸浮細(xì)胞,置于37℃含5%CO_2的細(xì)胞培養(yǎng)箱中培養(yǎng)。培養(yǎng)48小時后半量換液,棄去不貼壁的細(xì)胞。以后每2～3天換液一次,當(dāng)細(xì)胞生長達(dá)到90%融合時按1:3行傳代培養(yǎng)。(1)取2代MSCs在融合達(dá)到90%時,以10 mmol/L的MT為誘導(dǎo)劑誘導(dǎo)大鼠MSCs向神經(jīng)元樣細(xì)胞分化。分別在誘導(dǎo)后3,7,10,14d在倒置顯微鏡下觀察細(xì)胞形態(tài),以免疫熒光法鑒定神經(jīng)元特異性烯醇化酶(NSE)、神經(jīng)元細(xì)胞特異性標(biāo)志神經(jīng)絲蛋白(NF)、膠質(zhì)纖維酸性蛋白(GFAP)的表達(dá)。(2)在2代MSCs在融合達(dá)到90%時,以10 mmol/L MT及OECs上清液為誘導(dǎo)劑誘導(dǎo)MSCs向神經(jīng)元樣細(xì)胞分化,在倒置顯微鏡下觀察細(xì)胞形態(tài),在誘導(dǎo)后14天以免疫熒光法鑒定神經(jīng)元特異性烯醇化酶(NSE)、神經(jīng)元細(xì)胞特異性標(biāo)志神經(jīng)絲蛋白(NF)、膠質(zhì)纖維酸性蛋白(GFAP)的表達(dá),并使用westernblot法對上述指標(biāo)進(jìn)行定量分析。最后統(tǒng)計分析各組間結(jié)果有無差異。 結(jié)果在誘導(dǎo)前,大鼠MSCs以長梭形為主,有些呈圓形、多角形,少數(shù)有短小突起。(1)在以MT誘導(dǎo)3d后,見部分細(xì)胞胞體收縮,折光性增強(qiáng),伸出突起,部分胞體收縮成三角形或成為簡單的雙極或多極細(xì)胞。7d后較長的突起末端形成生長錐樣的膨大,呈現(xiàn)神經(jīng)元樣的形態(tài)。10d后神經(jīng)元樣細(xì)胞數(shù)量增多,細(xì)胞突起延長。14d后,神經(jīng)元樣的細(xì)胞數(shù)量進(jìn)一步增多,且?guī)讉�細(xì)胞的突起之間互相連接成網(wǎng)狀。繡導(dǎo)3d細(xì)胞開始表達(dá)nestin,7～14d表達(dá)nestin和NF,細(xì)胞不表達(dá)GFAP。(2)在以MT及OECs為誘導(dǎo)劑誘導(dǎo)3d后MSCs逐漸向神經(jīng)元樣細(xì)胞分化,呈現(xiàn)神經(jīng)元細(xì)胞樣形態(tài),在誘導(dǎo)14d后免疫熒光法鑒定NF、NSE均為陽性表達(dá),GFAP為陰性表達(dá)。MT及OECs上清液聯(lián)合誘導(dǎo)組變化較為顯著,westernblot法檢測亦可證實。 結(jié)論(1)MT可誘導(dǎo)MSCs向神經(jīng)元樣細(xì)胞分化,分化過程是:MSCs→神經(jīng)干樣細(xì)胞→神經(jīng)元樣細(xì)胞。(2)MT及OECs上清液可誘導(dǎo)大鼠MSCs分化為神經(jīng)元樣細(xì)胞,且二者聯(lián)合誘導(dǎo)效果優(yōu)于單獨誘導(dǎo)。
[Abstract]:Objective to study the differentiation of rat bone marrow mesenchymal stem cells (Marrow Mesenchymal Stem cells cells) into neuron-like cells induced by melatonin in vitro and olfactory ensheathing cells (OECs) supernatant.To investigate the differentiation of MSCs into neuroblast-like cells in vitro and the advantages and disadvantages of MTOECs supernatant induced by MT in vitro, and to find out the optimal induction scheme.It provides some experimental basis for studying the differentiation of MSCs into neuron-like cells and provides another kind of seed cells for cell transplantation.Methods the bone marrow tissue of healthy male Sprague-Dawley rats was collected under aseptic condition. The nucleated cell layer was obtained by density gradient centrifugation of Ficoll human lymphocyte isolate with density of 1.077g/mL. The nucleated cell layer was seeded into 25ml cell culture flask with a density of 5 脳 10~6/cm~2.Suspension cells were cultured on L-DMEM medium containing 10% fetal bovine serum in 37 鈩，

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