【摘要】：在中樞神經(jīng)系統(tǒng)中,神經(jīng)干細胞(Neural stem cells, NSCs)的增殖和分化調(diào)控機制在神經(jīng)發(fā)生(neurogenesis)過程中起著非常重要的作用,是當(dāng)今神經(jīng)發(fā)育生物學(xué)的重要研究內(nèi)容。神經(jīng)干細胞是一類具有自我更新以及分化成為神經(jīng)元、星形膠質(zhì)細胞和少突膠質(zhì)細胞的多潛能未分化干細胞,利用神經(jīng)干細胞不僅可以探討神經(jīng)系統(tǒng)發(fā)育的分子機理,也可作為一種替代手段用于中樞神經(jīng)損傷、退行性疾病和腦腫瘤等疾病的治療。因此,建立穩(wěn)定、可靠的NSCs體外培養(yǎng)模型,是探索神經(jīng)系統(tǒng)發(fā)育的機制和進行神經(jīng)干細胞體內(nèi)移植的基礎(chǔ),也是當(dāng)代神經(jīng)科學(xué)領(lǐng)域的研究熱點之一。 miRNA是一組非編碼的單鏈小RNA,平均長度22nt,在生物發(fā)育和細胞分化中發(fā)揮重要作用。miRNA的表達具有顯著的時序性和組織特異性。在動物中,miRNA5'端和靶分子mRNA3'UTR序列互補結(jié)合后,靶mRNA的翻譯起始后的表達受到抑制。近年來,越來越多的證據(jù)表明miRNA在神經(jīng)干細胞的自我更新和分化過程中起到了非常重要的作用。已有文獻報道,miR-9,124在大腦皮質(zhì)處均有特異性的表達,參與了神經(jīng)干細胞的分化過程,本室在前期工作中通過原位雜交技術(shù)篩選出一組在神經(jīng)系統(tǒng)發(fā)育的不同時期以及不同部位有特異性表達的miRNAs,提示我們這些miRNAs在NSCs增殖和分化過程中也可能介入了調(diào)控。為了更加深入的研究NSCs的調(diào)控機制,本文首先建立了小鼠神經(jīng)干細胞原代培養(yǎng)及其增殖和分化技術(shù)平臺：小鼠神經(jīng)干細胞(mNSCs)通過將胎齡為14.5-16.5天的胎鼠前腦皮層吹散分離獲得。經(jīng)過培養(yǎng)可使mNSCs成功增殖形成神經(jīng)球(Neurosphere)。將神經(jīng)球通過Accutase酶消化打散并經(jīng)過誘導(dǎo)分化,可使mNSCs成功分化成神經(jīng)元、星形膠質(zhì)細胞及少突膠質(zhì)細胞。然后我們提取分化前mNSCs和經(jīng)全反式維甲酸(RA)誘導(dǎo)3d后細胞的總RNA,通過Realtime-PCR方法我們發(fā)現(xiàn)一組miRNAs的表達量發(fā)生了明顯改變,其中miRNA-214變化量非常明顯,已有文獻報道m(xù)iRNA-214在神經(jīng)母細胞瘤分化,皮層發(fā)育,胚胎干細胞分化以及神經(jīng)突起的生長中都起到了非常重要的作用,我們通過TargetScan對miRNA-214可能結(jié)合的下游靶基因進行生物信息學(xué)分析,發(fā)現(xiàn)miRNA-214作用的靶基因包括參與了mNSCs自我更新和增殖過程的Nestin,Smad4等,提示我們miRNA-214可能具有抑制mNSCs自我更新和增殖而促進其分化的功能。因此,我們重點選取了miR-214作為研究對象,借助于新一代脂質(zhì)體轉(zhuǎn)染技術(shù)將miRNA-214過表達雙鏈模擬物或其抑制物高效瞬時轉(zhuǎn)染到mNSCs中,Western-Blot實驗證明miRNA-214的表達量降低后,神經(jīng)元特異性標(biāo)志蛋白β-tubulinⅢ在神經(jīng)干細胞分化過程中表達量減少,通過BrdU實驗發(fā)現(xiàn)過表達miRNA-214后神經(jīng)干細胞的增殖能力有所下降,免疫熒光實驗則發(fā)現(xiàn)miR-214能夠促進神經(jīng)干細胞向神經(jīng)元方向分化,從而進一步證明了miR-214在神經(jīng)干細胞的增殖和分化過程中的確起到了一定的作用。 上述工作以小鼠神經(jīng)干細胞技術(shù)平臺為模型,通過瞬時轉(zhuǎn)染的方法重點探索了miRNA-214在神經(jīng)干細胞增殖與分化過程中的功能,取得了一些初步結(jié)果,為其以后深入研究miRNA與下游靶基因相互作用的分子機制奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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