短期撤除bFGF、EGF的培养条件对神经干细胞增殖和分化的影响

发布时间：2018-01-01 07:17

本文关键词：短期撤除bFGF、EGF的培养条件对神经干细胞增殖和分化的影响　出处：《吉林大学》2012年硕士论文　论文类型：学位论文

【摘要】：表皮细胞生长因子(EGF)在人体的各种组织和体液内广泛存在，并对多种组织细胞具有强烈的促分裂作用。体外实验证明EGF在发育晚期促使神经前体细胞分裂增殖和分化，且促分化主要对胶原祖细胞起作用。成纤维细胞生长因子(FGF)是一种能够促进成纤维细胞生长的活性物质。碱性成纤维细胞生长因子(FGF2/bFGF)作为成纤维细胞生长因子家族成员之一，在胚胎早期发挥促进神经干细胞有丝分裂的作用，并维持其生存，且对胶质细胞和神经元的祖细胞都有促进分化的作用。因此，EGF与bFGF是神经干细胞体外培养体系中不可缺少的神经营养因子。此外神经干细胞自身还可产生多种调控信号对其增殖和分化进行调节，在中枢神经系统发育过程中Notch信号通路也对神经元分化的数量起着至关重要的作用。本研究通过神经干细胞培养技术、二次克隆技术、免疫组化技术、RT-PCR技术，检测短时撤除EGF、bFGF因子对神经干细胞增殖及分化的影响。实验方法：无菌提取孕14天胎鼠大脑皮质细胞，原代悬浮培养3天后，取一部分胰酶消化为单细胞，撤除EGF、bFGF，观察神经球克隆的数量。将神经球和消化为单细胞的神经干细胞贴壁生长，撤除EGF、bFGF，培养3天后进行PI/Hochest染色，比较撤除因子的神经干细胞与正常培养的神经干细胞之间的存活数量的差别。分别对培养3天和7天的神经球进行NF染色，比较两组神经干细胞的分化情况，并对培养3天及7天的神经球进行RT-PCR半定量检测神经元及胶质细胞的分化比例。实验结果显示：分离培养的大鼠神经干细胞悬浮培养3天后，光镜下显示细胞呈圆形球状的细胞团，并呈nestin阳性。经PI/Hochest染色后，对照组生存率为92%，撤除因子组生存率为75%，两组比较存在显著差异(P0.05)克隆后计数结果显示撤除因子组神经干细胞球的数量明显少于对照组(P0.05)。NF染色，撤除因子组神经元分化的数量明显多于对照组，，经RT-PCR检测分析结果支持免疫组化染色结果(P0.05)。此结果说明，在神经干细胞培养体系中撤除EGF、bFGF会影响神经干细胞的增殖，并促进神经干细胞的分化，尤其是神经干细胞向神经元方向的分化。随着对EGF、bFGF在神经细胞分化和增殖中的影响的进一步研究，期待神经干细胞增殖和分化的机制更加清晰明确，进而推进临床应用干细胞移植治疗各种神经退行性疾病的进程，使干细胞应用的前景更加广阔。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329.21

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