体外诱导人脐血间充质干细胞向多巴胺能神经元分化的研究

发布时间：2018-05-20 08:02

本文选题：间充质干细胞 + 神经元样细胞　；参考：《河北联合大学》2011年硕士论文

【摘要】：目的建立人脐血间充质干细胞(human umbilical blood-derived mesenchymal stem cells,HUCB-MSCs)分化为多巴胺能神经元的诱导体系,以明确其神经分化潜能,进而为临床利用细胞替代治疗帕金森病提供理论依据和实验基础。方法取第5代的HUCB-MSCs分别用三种不同的诱导方法向多巴胺能神经元样细胞诱导。即:抗氧化剂诱导法、神经营养因子诱导法、抗氧化剂联合神经营养因子诱导法。诱导期间观察细胞形态的变化。诱导结束后,用4%多聚甲醛固定细胞,免疫细胞化学法检测神经元特异性标志物Nestin、NSE、TH、DAT、DRD2和神经胶质细胞标志物GFAP的表达。并应用Real-time RT-PCR方法检测三种诱导方法向多巴胺能神经元发育的相关基因TH mRNA、DAT mRNA、DRD2 mRNA的表达。结果HUCB-MSCs在诱导分化前多数呈均一的长梭形的成纤维细胞样细胞,在经预诱导处理24h后,细胞体积缩小,立体感增强,边缘变得不规整,极少数细胞有细的突起。正式诱导后,细胞胞体进一步收缩,形成圆形、不规则的锥形、三角形,有的细胞有多个突起,而且发出分支,形成圆锥状终末端,有的突起逐渐伸长,终末端有类似神经元细胞的终结。双级或多极的突起互相连接呈网状。免疫细胞化学法检测显示三种诱导方法诱导后的细胞均表达多巴胺能神经元相关蛋白:TH、DAT、DRD2;Real-time RT-PCR检测显示对照组和诱导组均有TH mRNA、DAT mRNA、DRD2 mRNA表达。结论本实验选择HUCB-MSCs作为研究对象,通过体外培养传代、冻存、复苏,证明HUCB-MSCs可在体外稳定培养、扩增。HUCB-MSCs通过三种诱导方法均可分化为神经元样细胞和多巴胺能神经元样细胞,并表达多巴胺能神经元特异性标志物。表明HUCB-MSCs具有神经分化的潜能。
[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.
【学位授予单位】：河北联合大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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