本文關(guān)鍵詞：鼠胚神經(jīng)干細(xì)胞的體外培養(yǎng)及其定向分化的實(shí)驗(yàn)研究　出處：《南京醫(yī)科大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 神經(jīng)干細(xì)胞 定向分化 肝細(xì)胞生長因子 胰島素樣生長因子-1 腦源性神經(jīng)營養(yǎng)因子 bHLH基因

【摘要】：本研究旨在觀察肝細(xì)胞生長因子(HGF)、胰島素樣生長因子-1(IGF-1)和腦源性神經(jīng)營養(yǎng)因子(BDNF)對鼠胚神經(jīng)干細(xì)胞(neural stem cell，NSC)體外誘導(dǎo)分化的促進(jìn)作用，以及檢測內(nèi)源性bHLH基因-MASH1、neuroD、neurogenin2(Ngn2)在神經(jīng)干細(xì)胞分化過程中表達(dá)的動態(tài)變化，探索神經(jīng)干細(xì)胞分化的一些內(nèi)部分子機(jī)制。首先取孕14-17天的SD大鼠的胚胎腦組織，在體外分離培養(yǎng)得到神經(jīng)干細(xì)胞后，分為兩組，實(shí)驗(yàn)組以HGF、IGF-1、BDNF等作為誘導(dǎo)分化劑誘導(dǎo)其分化，而對照組僅加入胎牛血清(FBS)，以免疫組化的方法鑒定所獲得的神經(jīng)干細(xì)胞、神經(jīng)元(neuron)、星型膠質(zhì)細(xì)胞(gliocyte)和少突膠質(zhì)細(xì)胞(oliodendrocyte)，以鏡下直接計(jì)數(shù)和流式細(xì)胞儀的方法來計(jì)數(shù)誘導(dǎo)分化的神經(jīng)元比例。選取未分化、分化1天、3天、5天做為觀察和研究的時間段，采用RT-PCR的方法分析分化各個時期內(nèi)源性bHLH基因表達(dá)的差異。原代培養(yǎng)結(jié)果顯示：在培養(yǎng)基中生長的細(xì)胞不斷出現(xiàn)形態(tài)學(xué)的改變，，4天左右，由一個細(xì)胞分裂而來的細(xì)胞已經(jīng)長為幾十個細(xì)胞聚集在一起的懸浮生長的神經(jīng)克隆球，培養(yǎng)至1周左右時，神經(jīng)球增多、增大，邊緣可見到鋸齒狀的單細(xì)胞邊界，形態(tài)規(guī)則，立體感強(qiáng)。誘導(dǎo)分化4-8小時后，兩組在倒置顯微鏡下均可以觀察到部分原來呈懸浮生長的克隆球貼壁，逐漸失去其球狀的立體外觀。24小時后即可發(fā)現(xiàn)從細(xì)胞團(tuán)塊的邊緣向外長出突起，克隆球中的細(xì)胞有向外遷移的趨勢，3天后即有不同形態(tài)的細(xì)胞從克隆球周圍出現(xiàn)，5天后克隆球已完全附壁。Nestin是神經(jīng)
[Abstract]:The purpose of this study was to observe the hepatocyte growth factor (HGF), insulin-like growth factor -1 (IGF-1) and brain-derived neurotrophic factor (BDNF) on rat embryonic neural stem cells (neural stem cell, NSC) promotes differentiation in vitro, and to detect endogenous bHLH genes -MASH1, neuroD, neurogenin2 (Ngn2) in the dynamic changes of the expression of the differentiation of neural stem cells into neural stem, some internal molecular mechanism of cell differentiation. The brain tissue of SD rat embryos from the first 14-17 days of pregnancy, in isolated neural stem cells, divided into two groups, the experimental group with HGF, IGF-1 and BDNF as the inducer of differentiation inducing differentiation, while the control group only adding fetal bovine serum (FBS), stem cell and neuron method identified by immunohistochemistry for nerve (neuron), astrocytes (gliocyte) and oligodendrocytes (oliodendrocyte), the proportion of neurons through direct counting under microscope and flow cytometric methods to count differentiation. 1 days, 3 days and 5 days were selected as observation and research period. The difference of endogenous bHLH gene expression at different stages was analyzed by RT-PCR. The results showed that primary cultured in DMEM cells appeared morphological changes, about 4 days from a cell division and the cell has been long for dozens of cells together suspended growth neural clone ball, cultured for 1 weeks or so, neurospheres increased and enlarged, single the cell boundary, the edge can be seen jagged shape, three-dimensional sense of strong. After induction and differentiation for 4-8 hours, two groups could observe some of the original cloned spheres attached to the suspension growth under the inverted microscope, and gradually lost their spherical appearance. 24 hours later, it was found that protuberance grew outward from the edge of the cell mass, and the cells in the cloned sphere migrated outward. After 3 days, there were different forms of cells from the clones, and 5 days later, the clones were completely attached to the wall. Nestin is the nerve
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R329.2

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本文編號：1345198