本文關(guān)鍵詞：短期撤除bFGF、EGF的培養(yǎng)條件對神經(jīng)干細(xì)胞增殖和分化的影響　出處：《吉林大學(xué)》2012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 表皮細(xì)胞生長因子 成纖維細(xì)胞生長因子

【摘要】：表皮細(xì)胞生長因子(EGF)在人體的各種組織和體液內(nèi)廣泛存在，并對多種組織細(xì)胞具有強(qiáng)烈的促分裂作用。體外實(shí)驗(yàn)證明EGF在發(fā)育晚期促使神經(jīng)前體細(xì)胞分裂增殖和分化，且促分化主要對膠原祖細(xì)胞起作用。成纖維細(xì)胞生長因子(FGF)是一種能夠促進(jìn)成纖維細(xì)胞生長的活性物質(zhì)。堿性成纖維細(xì)胞生長因子(FGF2/bFGF)作為成纖維細(xì)胞生長因子家族成員之一，在胚胎早期發(fā)揮促進(jìn)神經(jīng)干細(xì)胞有絲分裂的作用，并維持其生存，且對膠質(zhì)細(xì)胞和神經(jīng)元的祖細(xì)胞都有促進(jìn)分化的作用。因此，EGF與bFGF是神經(jīng)干細(xì)胞體外培養(yǎng)體系中不可缺少的神經(jīng)營養(yǎng)因子。此外神經(jīng)干細(xì)胞自身還可產(chǎn)生多種調(diào)控信號對其增殖和分化進(jìn)行調(diào)節(jié)，在中樞神經(jīng)系統(tǒng)發(fā)育過程中Notch信號通路也對神經(jīng)元分化的數(shù)量起著至關(guān)重要的作用。 本研究通過神經(jīng)干細(xì)胞培養(yǎng)技術(shù)、二次克隆技術(shù)、免疫組化技術(shù)、RT-PCR技術(shù)，檢測短時撤除EGF、bFGF因子對神經(jīng)干細(xì)胞增殖及分化的影響。 實(shí)驗(yàn)方法：無菌提取孕14天胎鼠大腦皮質(zhì)細(xì)胞，原代懸浮培養(yǎng)3天后，取一部分胰酶消化為單細(xì)胞，撤除EGF、bFGF，觀察神經(jīng)球克隆的數(shù)量。將神經(jīng)球和消化為單細(xì)胞的神經(jīng)干細(xì)胞貼壁生長，撤除EGF、bFGF，培養(yǎng)3天后進(jìn)行PI/Hochest染色，比較撤除因子的神經(jīng)干細(xì)胞與正常培養(yǎng)的神經(jīng)干細(xì)胞之間的存活數(shù)量的差別。分別對培養(yǎng)3天和7天的神經(jīng)球進(jìn)行NF染色，比較兩組神經(jīng)干細(xì)胞的分化情況，并對培養(yǎng)3天及7天的神經(jīng)球進(jìn)行RT-PCR半定量檢測神經(jīng)元及膠質(zhì)細(xì)胞的分化比例。 實(shí)驗(yàn)結(jié)果顯示：分離培養(yǎng)的大鼠神經(jīng)干細(xì)胞懸浮培養(yǎng)3天后，光鏡下顯示細(xì)胞呈圓形球狀的細(xì)胞團(tuán)，并呈nestin陽性。經(jīng)PI/Hochest染色后，對照組生存率為92%，撤除因子組生存率為75%，兩組比較存在顯著差異(P0.05)克隆后計(jì)數(shù)結(jié)果顯示撤除因子組神經(jīng)干細(xì)胞球的數(shù)量明顯少于對照組(P0.05)。NF染色，撤除因子組神經(jīng)元分化的數(shù)量明顯多于對照組，，經(jīng)RT-PCR檢測分析結(jié)果支持免疫組化染色結(jié)果(P0.05)。此結(jié)果說明，在神經(jīng)干細(xì)胞培養(yǎng)體系中撤除EGF、bFGF會影響神經(jīng)干細(xì)胞的增殖，并促進(jìn)神經(jīng)干細(xì)胞的分化，尤其是神經(jīng)干細(xì)胞向神經(jīng)元方向的分化。 隨著對EGF、bFGF在神經(jīng)細(xì)胞分化和增殖中的影響的進(jìn)一步研究，期待神經(jīng)干細(xì)胞增殖和分化的機(jī)制更加清晰明確，進(jìn)而推進(jìn)臨床應(yīng)用干細(xì)胞移植治療各種神經(jīng)退行性疾病的進(jìn)程，使干細(xì)胞應(yīng)用的前景更加廣闊。
[Abstract]:Epidermal growth factor (EGF) is widely found in various tissues and body fluids of human body. In vitro experiments showed that EGF promoted the proliferation and differentiation of neural precursor cells in the late stage of development. Fibroblast growth factor (FGFs) is a kind of active substance that can promote the growth of fibroblasts. Basic fibroblast growth factor (bFGF). FGF2 / bFGFs are members of the fibroblast growth factor family. In the early embryonic stage, it can promote the mitosis of neural stem cells and maintain their survival, and promote the differentiation of glial cells and neuronal progenitor cells. EGF and bFGF are indispensable neurotrophic factors in the culture system of neural stem cells in vitro. In addition, neural stem cells can also produce a variety of regulatory signals to regulate their proliferation and differentiation. The Notch signaling pathway also plays an important role in the number of neuronal differentiation during the development of the central nervous system. In this study, neural stem cell culture, secondary cloning, immunohistochemistry and RT-PCR were used to detect the removal of EGF in a short time. Effect of bFGF factor on proliferation and differentiation of neural stem cells. Methods: fetal rat cerebral cortex cells were extracted by aseptic method. After suspension culture for 3 days, some trypsin was digested into single cells and EGF- bFGF was removed. The number of neuronal clones was observed. The ball and the neural stem cells digested into single cells were adhered to the wall, and EGFN bFGF was removed. After 3 days of culture, the cells were stained with PI/Hochest. The survival number of neural stem cells (NSCs) which were removed factor was compared with that of normal cultured neural stem cells (NSCs). NF staining was performed on the neurons cultured for 3 and 7 days, respectively, and the differentiation of neural stem cells was compared between the two groups. The differentiation ratio of neuronal and glial cells was measured by RT-PCR on the 3rd and 7th day of culture. The results showed that after 3 days of suspension culture of rat neural stem cells, the cells were round globular cells under light microscope. After PI/Hochest staining, the survival rate of the control group was 92% and that of the removal group was 75%. There was significant difference between the two groups (P0.05) after cloning the results showed that the number of neural stem cell balls in the removal factor group was significantly less than that in the control group. The number of neuronal differentiation in the withdrawal group was significantly higher than that in the control group. The results of RT-PCR analysis supported the immunohistochemical staining results (P 0.05). The removal of EGF- bFGF from the culture system of neural stem cells will affect the proliferation of neural stem cells and promote the differentiation of neural stem cells, especially the differentiation of neural stem cells into neurons. With the further study of the effect of EGF- bFGF on neural cell differentiation and proliferation, it is expected that the mechanism of neural stem cell proliferation and differentiation will be more clear. Further, the clinical application of stem cell transplantation in the treatment of various neurodegenerative diseases progress, making stem cell application more promising.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329.21

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 孫晉浩,楊琳,高英茂;表皮生長因子促進(jìn)胚胎神經(jīng)干細(xì)胞生長分化的研究[J];山東大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2004年01期

2 游曉青,張琦,林玲,胡建石,姜芬,鄭志z

本文編號：1363472