本文選題：間充質(zhì)干細(xì)胞 + 神經(jīng)元樣細(xì)胞�。� 參考：《河北聯(lián)合大學(xué)》2011年碩士論文

【摘要】：目的建立人臍血間充質(zhì)干細(xì)胞(human umbilical blood-derived mesenchymal stem cells,HUCB-MSCs)分化為多巴胺能神經(jīng)元的誘導(dǎo)體系,以明確其神經(jīng)分化潛能,進(jìn)而為臨床利用細(xì)胞替代治療帕金森病提供理論依據(jù)和實(shí)驗(yàn)基礎(chǔ)。 方法取第5代的HUCB-MSCs分別用三種不同的誘導(dǎo)方法向多巴胺能神經(jīng)元樣細(xì)胞誘導(dǎo)。即:抗氧化劑誘導(dǎo)法、神經(jīng)營(yíng)養(yǎng)因子誘導(dǎo)法、抗氧化劑聯(lián)合神經(jīng)營(yíng)養(yǎng)因子誘導(dǎo)法。誘導(dǎo)期間觀察細(xì)胞形態(tài)的變化。誘導(dǎo)結(jié)束后,用4%多聚甲醛固定細(xì)胞,免疫細(xì)胞化學(xué)法檢測(cè)神經(jīng)元特異性標(biāo)志物Nestin、NSE、TH、DAT、DRD2和神經(jīng)膠質(zhì)細(xì)胞標(biāo)志物GFAP的表達(dá)。并應(yīng)用Real-time RT-PCR方法檢測(cè)三種誘導(dǎo)方法向多巴胺能神經(jīng)元發(fā)育的相關(guān)基因TH mRNA、DAT mRNA、DRD2 mRNA的表達(dá)。 結(jié)果HUCB-MSCs在誘導(dǎo)分化前多數(shù)呈均一的長(zhǎng)梭形的成纖維細(xì)胞樣細(xì)胞,在經(jīng)預(yù)誘導(dǎo)處理24h后,細(xì)胞體積縮小,立體感增強(qiáng),邊緣變得不規(guī)整,極少數(shù)細(xì)胞有細(xì)的突起。正式誘導(dǎo)后,細(xì)胞胞體進(jìn)一步收縮,形成圓形、不規(guī)則的錐形、三角形,有的細(xì)胞有多個(gè)突起,而且發(fā)出分支,形成圓錐狀終末端,有的突起逐漸伸長(zhǎng),終末端有類似神經(jīng)元細(xì)胞的終結(jié)。雙級(jí)或多極的突起互相連接呈網(wǎng)狀。免疫細(xì)胞化學(xué)法檢測(cè)顯示三種誘導(dǎo)方法誘導(dǎo)后的細(xì)胞均表達(dá)多巴胺能神經(jīng)元相關(guān)蛋白:TH、DAT、DRD2;Real-time RT-PCR檢測(cè)顯示對(duì)照組和誘導(dǎo)組均有TH mRNA、DAT mRNA、DRD2 mRNA表達(dá)。 結(jié)論本實(shí)驗(yàn)選擇HUCB-MSCs作為研究對(duì)象,通過(guò)體外培養(yǎng)傳代、凍存、復(fù)蘇,證明HUCB-MSCs可在體外穩(wěn)定培養(yǎng)、擴(kuò)增。HUCB-MSCs通過(guò)三種誘導(dǎo)方法均可分化為神經(jīng)元樣細(xì)胞和多巴胺能神經(jīng)元樣細(xì)胞,并表達(dá)多巴胺能神經(jīng)元特異性標(biāo)志物。表明HUCB-MSCs具有神經(jīng)分化的潛能。
[Abstract]:Objective to establish an induction system for differentiation of human umbilical blood-derived mesenchymal stem cells into dopaminergic neurons from human umbilical cord blood mesenchymal stem cells (HUCB-MSCs), so as to clarify its neural differentiation potential and to provide theoretical and experimental basis for clinical treatment of Parkinson's disease with cell replacement therapy. Methods the fifth passage of HUCB-MSCs was induced to dopaminergic neuron-like cells by three different induction methods. Namely: antioxidant induction method, neurotrophic factor induction method, antioxidant combined with neurotrophic factor induction method. Cell morphology was observed during induction. After induction, 4% paraformaldehyde was used to immobilize the cells, and immunocytochemistry was used to detect the expression of neuron-specific marker Nestinine (Nestinine) and neuroglial marker (GFAP). Real-time RT-PCR method was used to detect the expression of DRD2 mRNA, a gene associated with the development of dopaminergic neurons (TH mRNA-DAT mRNA-DRD2 mRNA). Results most of the long fusiform fibroblast-like cells in HUCB-MSCs before differentiation were reduced in volume, increased in stereosensitivity and irregular in edge, and a few cells had fine protrusions after 24 hours of pre-induction. After formal induction, the cell body shrinks further, forming round, irregular cones, triangles, and some cells have multiple protrusions, and branch, forming conical ends, and some processes gradually elongate. Terminal has similar neuronal cell termination. Double or multipolar processes are connected to each other in a reticular form. The expression of dopaminergic neuron-associated protein: TH-mRNA-DAT mRNA-DRD2 mRNA was detected by immunocytochemistry and real-time RT-PCR analysis of dopaminergic neuron-associated protein: TH-mRNA-DAT mRNA-DRD2 mRNA in both the control group and the induced group. Conclusion in this experiment, HUCB-MSCs was selected as the research object. It was proved that HUCB-MSCs could be cultured stably in vitro through passage, cryopreservation and resuscitation in vitro, and HUCB-MSCs could be differentiated into neuron-like cells and dopaminergic neuron-like cells by three induction methods. The specific markers of dopaminergic neurons were also expressed. It is suggested that HUCB-MSCs has the potential of neural differentiation.
【學(xué)位授予單位】：河北聯(lián)合大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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本文編號(hào)：1913810