【摘要】：目的：谷氨酸能神經(jīng)元作為中樞神經(jīng)系統(tǒng)中最重要的興奮性神經(jīng)元,調(diào)節(jié)著釋放進(jìn)入突觸間隙谷氨酸遞質(zhì)的量,參與了幾乎所有腦的功能。它的異常和丟失會(huì)導(dǎo)致興奮性神經(jīng)遞質(zhì)谷氨酸的異常,從而誘發(fā)多種神經(jīng)系統(tǒng)疾病,諸如阿爾茲海默病(Alzheimer's disease,AD)、帕金森病(Parkinson's disease, PD)、精神分裂癥、抑郁癥、癲癇、耳聾發(fā)病等。而胚胎干細(xì)胞(Embryonic stem cells, ESCs)以其可體外無(wú)限增殖和全能性等生物學(xué)特性,有望為神經(jīng)系統(tǒng)疾病細(xì)胞替代性治療提供細(xì)胞來(lái)源。以往的研究都集中于胚胎干細(xì)胞經(jīng)外部因素的誘導(dǎo)而轉(zhuǎn)化為神經(jīng)元細(xì)胞,不僅方法過(guò)于復(fù)雜,而且誘導(dǎo)效率也較低,因此本論文擬開(kāi)展研究一種新的胚胎干細(xì)胞神經(jīng)分化的誘導(dǎo)方法。方法：1.分離BALB/c小鼠囊胚內(nèi)細(xì)胞團(tuán)(Inner cell mass, ICM),放置于飼養(yǎng)層細(xì)胞上生長(zhǎng),制備小鼠胚胎干細(xì)胞(mouse Embryonic stem cells, mESCs),培養(yǎng)傳代并鑒定后,凍存?zhèn)溆谩?.利用優(yōu)化的無(wú)血清懸浮培養(yǎng)法誘導(dǎo)mESCs向端腦前體細(xì)胞選擇分化,并用流式細(xì)胞儀檢測(cè)Bfl+和Emx1+端腦皮質(zhì)前體細(xì)胞的比例。3.PCR擴(kuò)增不同分化階段細(xì)胞標(biāo)志基因胚胎干細(xì)胞關(guān)鍵蛋白(Oct3/4)、巢蛋白(Nestin)、β-微管蛋白Ⅲ(Tuj1)、膠質(zhì)纖維酸性蛋白(GFAP)的cDNA,瓊脂糖凝膠電泳檢測(cè)其含量。4.環(huán)巴胺誘導(dǎo)端腦前體細(xì)胞選擇分化為VGLUT1谷氨酸能神經(jīng)元。5.免疫熒光染色優(yōu)化法誘導(dǎo)所得細(xì)胞的谷氨酸能神經(jīng)元標(biāo)記Tujl+和VGlUT1+。6.膜片鉗記錄檢測(cè)體內(nèi)、外VGLUT1神經(jīng)元電生理特性。結(jié)果：1)該法可使mESCs自主發(fā)生神經(jīng)細(xì)胞分化,主要依賴(lài)細(xì)胞的內(nèi)部機(jī)制和弱的內(nèi)源性細(xì)胞外信號(hào)。2)此種誘導(dǎo)技術(shù)能使mESCs高效分化為Bfl+端腦前體細(xì)胞,達(dá)總細(xì)胞量的70%,其中89%細(xì)胞表達(dá)皮質(zhì)標(biāo)記物Emx1。3)該法能使mESCs按照一個(gè)先神經(jīng)元-后膠質(zhì)細(xì)胞的順序有效分化,與體內(nèi)胚胎神經(jīng)組織發(fā)育先后順序一致。4)環(huán)巴胺能提高生成VGLUT1+谷氨酸能神經(jīng)元的比例,達(dá)總細(xì)胞量的70%。5)該法誘導(dǎo)所得的端腦前體細(xì)胞可分化為有功能的皮質(zhì)谷氨酸能神經(jīng)元。結(jié)論：通過(guò)優(yōu)化無(wú)血清懸浮培養(yǎng)技術(shù),我們成功建立了一種高效誘導(dǎo)胚胎干細(xì)胞分化為大腦皮質(zhì)谷氨酸能神經(jīng)元的技術(shù),這不僅為治療和研究神經(jīng)組織退化性疾病或腦損傷性疾病提供了潛在的細(xì)胞來(lái)源,而且對(duì)于了解神經(jīng)組織的發(fā)育機(jī)制也具有一定的科學(xué)意義。
[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，

本文編號(hào)：2239085