胚胎干细胞诱导分化为大脑皮质谷氨酸能神经元的实验研究
发布时间：2018-09-12 13:03
【摘要】：目的：谷氨酸能神经元作为中枢神经系统中最重要的兴奋性神经元,调节着释放进入突触间隙谷氨酸递质的量,参与了几乎所有脑的功能。它的异常和丢失会导致兴奋性神经递质谷氨酸的异常,从而诱发多种神经系统疾病,诸如阿尔兹海默病(Alzheimer's disease,AD)、帕金森病(Parkinson's disease, PD)、精神分裂症、抑郁症、癫痫、耳聋发病等。而胚胎干细胞(Embryonic stem cells, ESCs)以其可体外无限增殖和全能性等生物学特性,有望为神经系统疾病细胞替代性治疗提供细胞来源。以往的研究都集中于胚胎干细胞经外部因素的诱导而转化为神经元细胞,不仅方法过于复杂,而且诱导效率也较低,因此本论文拟开展研究一种新的胚胎干细胞神经分化的诱导方法。方法：1.分离BALB/c小鼠囊胚内细胞团(Inner cell mass, ICM),放置于饲养层细胞上生长,制备小鼠胚胎干细胞(mouse Embryonic stem cells, mESCs),培养传代并鉴定后,冻存备用。2.利用优化的无血清悬浮培养法诱导mESCs向端脑前体细胞选择分化,并用流式细胞仪检测Bfl+和Emx1+端脑皮质前体细胞的比例。3.PCR扩增不同分化阶段细胞标志基因胚胎干细胞关键蛋白(Oct3/4)、巢蛋白(Nestin)、β-微管蛋白Ⅲ(Tuj1)、胶质纤维酸性蛋白(GFAP)的cDNA,琼脂糖凝胶电泳检测其含量。4.环巴胺诱导端脑前体细胞选择分化为VGLUT1谷氨酸能神经元。5.免疫荧光染色优化法诱导所得细胞的谷氨酸能神经元标记Tujl+和VGlUT1+。6.膜片钳记录检测体内、外VGLUT1神经元电生理特性。结果：1)该法可使mESCs自主发生神经细胞分化,主要依赖细胞的内部机制和弱的内源性细胞外信号。2)此种诱导技术能使mESCs高效分化为Bfl+端脑前体细胞,达总细胞量的70%,其中89%细胞表达皮质标记物Emx1。3)该法能使mESCs按照一个先神经元-后胶质细胞的顺序有效分化,与体内胚胎神经组织发育先后顺序一致。4)环巴胺能提高生成VGLUT1+谷氨酸能神经元的比例,达总细胞量的70%。5)该法诱导所得的端脑前体细胞可分化为有功能的皮质谷氨酸能神经元。结论：通过优化无血清悬浮培养技术,我们成功建立了一种高效诱导胚胎干细胞分化为大脑皮质谷氨酸能神经元的技术,这不仅为治疗和研究神经组织退化性疾病或脑损伤性疾病提供了潜在的细胞来源,而且对于了解神经组织的发育机制也具有一定的科学意义。
[Abstract]:Aim: as the most important excitatory neurons in the central nervous system glutaminergic neurons regulate the release of glutamate transmitters into the synaptic space and participate in almost all brain functions. Its abnormality and loss can lead to the abnormality of excitatory neurotransmitter glutamate, which can induce many nervous system diseases, such as Alzheimer's disease (Alzheimer's disease,AD), Parkinson's disease (Parkinson's disease, PD),) schizophrenia, depression, epilepsy, deafness and so on. However, embryonic stem cell (Embryonic stem cells, ESCs), with its biological characteristics of infinite proliferation and totipotency in vitro, is expected to provide a cell source for alternative treatment of nervous system diseases. Previous studies have focused on the transformation of embryonic stem cells into neuronal cells induced by external factors. Therefore, this paper intends to develop a new method for inducing neural differentiation of embryonic stem cells. Method 1: 1. BALB/c mouse blastocyst (Inner cell mass, ICM), was isolated from blastocyst and placed on feeder layer cells to grow. Mouse embryonic stem cells (mouse Embryonic stem cells, mESCs),) were prepared and passaged and identified after cryopreservation. The optimal serum-free suspension culture method was used to induce the selective differentiation of mESCs into telencephalic progenitor cells. Flow cytometry was used to detect the ratio of Bfl and Emx1 terminal cerebral cortex precursor cells. 3. PCR amplification of cell marker gene embryonic stem cell key protein (Oct3/4), nestin (Nestin), 尾 -tubulin 鈪，
本文编号：2239085
[Abstract]:Aim: as the most important excitatory neurons in the central nervous system glutaminergic neurons regulate the release of glutamate transmitters into the synaptic space and participate in almost all brain functions. Its abnormality and loss can lead to the abnormality of excitatory neurotransmitter glutamate, which can induce many nervous system diseases, such as Alzheimer's disease (Alzheimer's disease,AD), Parkinson's disease (Parkinson's disease, PD),) schizophrenia, depression, epilepsy, deafness and so on. However, embryonic stem cell (Embryonic stem cells, ESCs), with its biological characteristics of infinite proliferation and totipotency in vitro, is expected to provide a cell source for alternative treatment of nervous system diseases. Previous studies have focused on the transformation of embryonic stem cells into neuronal cells induced by external factors. Therefore, this paper intends to develop a new method for inducing neural differentiation of embryonic stem cells. Method 1: 1. BALB/c mouse blastocyst (Inner cell mass, ICM), was isolated from blastocyst and placed on feeder layer cells to grow. Mouse embryonic stem cells (mouse Embryonic stem cells, mESCs),) were prepared and passaged and identified after cryopreservation. The optimal serum-free suspension culture method was used to induce the selective differentiation of mESCs into telencephalic progenitor cells. Flow cytometry was used to detect the ratio of Bfl and Emx1 terminal cerebral cortex precursor cells. 3. PCR amplification of cell marker gene embryonic stem cell key protein (Oct3/4), nestin (Nestin), 尾 -tubulin 鈪，
本文编号：2239085
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