MicroRNA-214在小鼠神经干细胞增殖和分化过程中功能的初步研究

发布时间：2018-08-28 19:55

【摘要】：在中枢神经系统中,神经干细胞(Neural stem cells, NSCs)的增殖和分化调控机制在神经发生(neurogenesis)过程中起着非常重要的作用,是当今神经发育生物学的重要研究内容。神经干细胞是一类具有自我更新以及分化成为神经元、星形胶质细胞和少突胶质细胞的多潜能未分化干细胞,利用神经干细胞不仅可以探讨神经系统发育的分子机理,也可作为一种替代手段用于中枢神经损伤、退行性疾病和脑肿瘤等疾病的治疗。因此,建立稳定、可靠的NSCs体外培养模型,是探索神经系统发育的机制和进行神经干细胞体内移植的基础,也是当代神经科学领域的研究热点之一。 miRNA是一组非编码的单链小RNA,平均长度22nt,在生物发育和细胞分化中发挥重要作用。miRNA的表达具有显著的时序性和组织特异性。在动物中,miRNA5'端和靶分子mRNA3'UTR序列互补结合后,靶mRNA的翻译起始后的表达受到抑制。近年来,越来越多的证据表明miRNA在神经干细胞的自我更新和分化过程中起到了非常重要的作用。已有文献报道,miR-9,124在大脑皮质处均有特异性的表达,参与了神经干细胞的分化过程,本室在前期工作中通过原位杂交技术筛选出一组在神经系统发育的不同时期以及不同部位有特异性表达的miRNAs,提示我们这些miRNAs在NSCs增殖和分化过程中也可能介入了调控。为了更加深入的研究NSCs的调控机制,本文首先建立了小鼠神经干细胞原代培养及其增殖和分化技术平台：小鼠神经干细胞(mNSCs)通过将胎龄为14.5-16.5天的胎鼠前脑皮层吹散分离获得。经过培养可使mNSCs成功增殖形成神经球(Neurosphere)。将神经球通过Accutase酶消化打散并经过诱导分化,可使mNSCs成功分化成神经元、星形胶质细胞及少突胶质细胞。然后我们提取分化前mNSCs和经全反式维甲酸(RA)诱导3d后细胞的总RNA,通过Realtime-PCR方法我们发现一组miRNAs的表达量发生了明显改变,其中miRNA-214变化量非常明显,已有文献报道miRNA-214在神经母细胞瘤分化,皮层发育,胚胎干细胞分化以及神经突起的生长中都起到了非常重要的作用,我们通过TargetScan对miRNA-214可能结合的下游靶基因进行生物信息学分析,发现miRNA-214作用的靶基因包括参与了mNSCs自我更新和增殖过程的Nestin,Smad4等,提示我们miRNA-214可能具有抑制mNSCs自我更新和增殖而促进其分化的功能。因此,我们重点选取了miR-214作为研究对象,借助于新一代脂质体转染技术将miRNA-214过表达双链模拟物或其抑制物高效瞬时转染到mNSCs中,Western-Blot实验证明miRNA-214的表达量降低后,神经元特异性标志蛋白β-tubulinⅢ在神经干细胞分化过程中表达量减少,通过BrdU实验发现过表达miRNA-214后神经干细胞的增殖能力有所下降,免疫荧光实验则发现miR-214能够促进神经干细胞向神经元方向分化,从而进一步证明了miR-214在神经干细胞的增殖和分化过程中的确起到了一定的作用。上述工作以小鼠神经干细胞技术平台为模型,通过瞬时转染的方法重点探索了miRNA-214在神经干细胞增殖与分化过程中的功能,取得了一些初步结果,为其以后深入研究miRNA与下游靶基因相互作用的分子机制奠定了基础。
[Abstract]:Neural stem cells (NSCs) play an important role in the regulation of proliferation and differentiation in the central nervous system (CNS) and play an important role in neurogenesis. NSCs are a class of neurons with self-renewal and differentiation into neurons and astrocytes. Neural stem cells can not only explore the molecular mechanism of nervous system development, but also be used as an alternative method for the treatment of central nervous system injury, degenerative diseases and brain tumors. The mechanism of systemic development and the basis of neural stem cell transplantation in vivo are also hot topics in the field of neuroscience.
MicroRNAs are a group of non-coding single-stranded small RNAs with an average length of 22 nt and play an important role in biological development and cell differentiation. The expression of microRNAs has significant timing and tissue specificity. Many evidences show that microRNAs play an important role in the self-renewal and differentiation of neural stem cells. It has been reported that microRNAs-9,124 are specifically expressed in the cerebral cortex and participate in the differentiation of neural stem cells. In our previous work, we screened a group of neurons in the nervous system by in situ hybridization. In order to further study the regulation mechanism of NSCs, we first established a mouse neural stem cell primary culture and its proliferation and differentiation technology platform: mouse neural stem fine. Cells (mNSCs) were isolated from the forebrain cortex of fetal mice aged 14.5-16.5 days. After culture, the mNSCs could successfully proliferate and form neurospheres (Neurospheres). After digestion and differentiation by Accutase, the neurospheres could be successfully differentiated into neurons, astrocytes and oligodendrocytes. Total RNA of cells 3 days after induction with all-trans retinoic acid (RA) and mNSCs before differentiation was obtained. Realtime-PCR showed that the expression of a group of microRNAs had changed significantly. Among them, the expression of microRNAs-214 was very obvious. It has been reported that microRNAs-214 were involved in neuroblastoma differentiation, cortical development, embryonic stem cell differentiation and neurite outgrowth. We analyzed the downstream target genes of microRNAs-214 by TargetScan. We found that the target genes of microRNAs-214 include Nestin, Smad4, which are involved in the self-renewal and proliferation of mNSCs, suggesting that microRNAs-214 may inhibit the self-renewal and proliferation of mNSCs. Therefore, we focused on the role of microRNA-214 in promoting differentiation. With the help of a new generation of liposome transfection technology, microRNA-214 over-expressed double-stranded mimics or their inhibitors were transfected into mNSCs efficiently and instantaneously. Western-Blot experiments showed that the expression of microRNA-214 decreased, the neuron-specific marker protein beta-tubuli. The expression of nIII decreased during the differentiation of neural stem cells. BrdU assay showed that the proliferation of neural stem cells decreased after overexpression of microRNA214. Immunofluorescence assay showed that microRNA214 could promote the differentiation of neural stem cells into neurons, which further proved that microRNA214 was involved in the proliferation and differentiation of neural stem cells. Indeed, it played a certain role.
Based on the mouse neural stem cell technology platform, the function of microRNA-214 in the proliferation and differentiation of neural stem cells was explored by transient transfection, and some preliminary results were obtained, which laid a foundation for further study of the molecular mechanism of interaction between microRNA and downstream target genes.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

【相似文献】