胚胎干细胞同步化处理、神经分化及Aβ对分化神经元NFκB通路的影响

发布时间：2018-08-28 12:39

【摘要】：胚胎干细胞(ESC)诱导分化为神经细胞的体外模型类似早期的胚胎发育模式,利用此模型,辅之以分子生物学、细胞生物学及免疫学等技术手段,可以有效进行神经发生分化及一些遗传性神经致病机制的研究。胚体(EBs)是ESC分化前形成的团聚体,在ESC神经分化过程中具有承前启后的作用,EBs内细胞的周期状态与诱导分化的关系未见报道。无血清培养方法诱导神经分化过程中,碱性成纤维生长因子(bFGF)是EBs贴壁后诱导分化阶段必须添加的生长因子,bFGF可能在ESC神经诱导分化阶段发挥神经营养作用,但由于bFGF能触发体外培养的皮层神经元释放兴奋性递质谷氨酸(Glu),故也可能在神经诱导分化阶段发挥神经刺激兴奋性的作用。利用ESC诱导分化为神经细胞的体外模型可以从神经发育学的角度分析阿尔茨海默症(AD)的发生机制,尤其是神经元从分化发生到凋亡或死亡的发育模式,为我们在体外研究β-淀粉样蛋白(Aβ)对不同年龄阶段神经元的影响提供了可能。首先,建立了基于本实验室条件下的ESC诱导分化神经元细胞方法体系。我们在Bain“4+/4-”RA诱导法的基础上,对小鼠D3系ESC采用“2+2”(即悬滴培养2天再悬浮培养两天)的培养方式形成EBs,全反式维甲酸(RA)诱导的ESC在体外分化为神经元样细胞,并初步分析了ESC诱导分化的神经元细胞生物学特性。结果发现,我们诱导ESC分化产生了谷氨酸能的神经元细胞,通过RT-PCR分析鉴定,这类神经元细胞表达了NMDA受体亚型;使用荧光分子探针标记发现,分化的神经元细胞能够被Glu刺激后开放NMDA受体通道,使胞内钙离子浓度升高,并被NMDA受体拮抗剂MK801阻断。其次,通过探索不同周期时相ESC对EBs形成的影响、不同增殖状态ESC对神经分化的影响等,确立ESC的细胞周期状态与EBs的结构形成的关系,发现血清饥饿同步化可以增强EBs的分化潜能性。通过血清饥饿、脱氧胸苷和秋水仙素阻断法分别有效地将ESC阻断在G0/G1、G1/S、G2/M相,研究了同步化后的细
[Abstract]:The model of embryonic stem cell (ESC) induced differentiation into neuronal cells in vitro is similar to the early embryonic development model, which is supplemented by molecular biology, cell biology and immunology, etc. It can effectively study the neurogenesis and differentiation and some genetic neuropathogenetic mechanisms. Embryonic (EBs) is an agglomerate formed before differentiation of ESC. The relationship between cell cycle state and induction of differentiation in ESC neural differentiation is not reported. In the process of nerve differentiation induced by serum-free culture, basic fibroblast growth factor (bFGF), which must be added in the stage of differentiation induced by EBs after adhesion, may play a neurotrophic role in the stage of nerve differentiation induced by ESC. However, bFGF can trigger the release of excitatory neurotransmitter glutamate (Glu), from cultured cortical neurons in vitro, so it may also play an excitatory role in neuronal induction and differentiation. The in vitro model of differentiation into nerve cells induced by ESC can be used to analyze the mechanism of (AD) in Alzheimer's disease from the perspective of neurogenesis, especially the developmental pattern of neurons from differentiation to apoptosis or death. It is possible for us to study the effect of 尾 -amyloid protein (A 尾) on neurons at different age stages in vitro. Firstly, the method system of ESC induced differentiation neuron cells was established under the condition of our laboratory. On the basis of Bain "4 / 4-" RA induction, we used "22" (2 days suspension culture) to form ESC induced by EBs, all-trans retinoic acid (RA) to differentiate into neuron-like cells in vitro. The biological characteristics of neuronal cells induced by ESC were preliminarily analyzed. The results showed that we induced ESC to differentiate into glutamatergic neuronal cells, which were identified by RT-PCR analysis and expressed NMDA receptor subtypes. The differentiated neurons were stimulated by Glu to open up NMDA receptor channels, which increased intracellular calcium concentration and were blocked by NMDA receptor antagonist MK801. Secondly, the relationship between the cell cycle state of ESC and the structure of EBs was established by exploring the effect of ESC on the formation of EBs and the effect of ESC on neural differentiation in different proliferative states. It was found that synchronization of serum starvation could enhance the differentiation potential of EBs. Serum starvation, deoxythymidine and colchicine blocking of ESC in G _ 0 / G _ 1 / S _ 1 / S _ 1 / G _ 2 / M phase were used to study the synchronized fineness.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R329.2

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