本文選題：骨髓間充質干細胞 + 褪黑素��； 參考：《江蘇大學》2009年碩士論文

【摘要】： 目的研究大鼠骨髓間充質干細胞(Marrow Mesenchymal Stem Cells,MSCs)經分離純化、體外培養(yǎng)后,以褪黑激素(Melatonin,MT)、嗅鞘細胞(OlfactoryEnsheathing Cells,OECs)上清液誘導MSCs向神經元樣細胞分化的情況。探討MT體外誘導MSCs不同時段向神經樣細胞分化的情況,以及MT、OECs上清液聯合誘導的優(yōu)劣,找出二者最優(yōu)誘導方案。為更好的研究MSCs向神經元樣細胞分化提供一定的實驗基礎,為細胞移植提供又一類種子細胞。 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人淋巴細胞分離液密度梯度離心法獲取有核細胞層,以5×10~6/cm~2密度接種于25ml細胞培養(yǎng)瓶,用含10%胎牛血清的L-DMEM培養(yǎng)基懸浮細胞,置于37℃含5%CO_2的細胞培養(yǎng)箱中培養(yǎng)。培養(yǎng)48小時后半量換液,棄去不貼壁的細胞。以后每2～3天換液一次,當細胞生長達到90%融合時按1:3行傳代培養(yǎng)。(1)取2代MSCs在融合達到90%時,以10 mmol/L的MT為誘導劑誘導大鼠MSCs向神經元樣細胞分化。分別在誘導后3,7,10,14d在倒置顯微鏡下觀察細胞形態(tài),以免疫熒光法鑒定神經元特異性烯醇化酶(NSE)、神經元細胞特異性標志神經絲蛋白(NF)、膠質纖維酸性蛋白(GFAP)的表達。(2)在2代MSCs在融合達到90%時,以10 mmol/L MT及OECs上清液為誘導劑誘導MSCs向神經元樣細胞分化,在倒置顯微鏡下觀察細胞形態(tài),在誘導后14天以免疫熒光法鑒定神經元特異性烯醇化酶(NSE)、神經元細胞特異性標志神經絲蛋白(NF)、膠質纖維酸性蛋白(GFAP)的表達,并使用westernblot法對上述指標進行定量分析。最后統(tǒng)計分析各組間結果有無差異。 結果在誘導前,大鼠MSCs以長梭形為主,有些呈圓形、多角形,少數有短小突起。(1)在以MT誘導3d后,見部分細胞胞體收縮,折光性增強,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胞。7d后較長的突起末端形成生長錐樣的膨大,呈現神經元樣的形態(tài)。10d后神經元樣細胞數量增多,細胞突起延長。14d后,神經元樣的細胞數量進一步增多,且?guī)讉�細胞的突起之間互相連接成網狀。繡導3d細胞開始表達nestin,7～14d表達nestin和NF,細胞不表達GFAP。(2)在以MT及OECs為誘導劑誘導3d后MSCs逐漸向神經元樣細胞分化,呈現神經元細胞樣形態(tài),在誘導14d后免疫熒光法鑒定NF、NSE均為陽性表達,GFAP為陰性表達。MT及OECs上清液聯合誘導組變化較為顯著,westernblot法檢測亦可證實。 結論(1)MT可誘導MSCs向神經元樣細胞分化,分化過程是:MSCs→神經干樣細胞→神經元樣細胞。(2)MT及OECs上清液可誘導大鼠MSCs分化為神經元樣細胞,且二者聯合誘導效果優(yōu)于單獨誘導。
[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，

本文編號：1753336