运用人诱导多能干细胞建立唐氏综合征中枢神经系统病变模型的研究

发布时间：2018-12-05 19:13

【摘要】：诱导多能干细胞(induced Pluripotent Stem Cell or iPSC)技术的产生和发展为建立神经系统疾病的体外细胞模型,研究个体特异的疾病发生机理提供了新的方法。然而,既往大多数关于神经系统疾病模型的研究主要局限于神经元的病变,通常忽略了胶质细胞在疾病表型中的作用。为了模拟唐氏综合症发病过程中细胞和分子水平上的缺陷,我们通过基因转染技术将人体皮肤成纤维细胞重新编程为诱导多能干细胞。其中有两株细胞系来源于唐氏综合症患者,另外两株细胞系来源于正常人作为对照。然后,经过体外培养,将诱导多能干细胞定向分化为神经干细胞,神经元及星形胶质细胞,从而建立唐氏综合症疾病模型。研究结果表明,相对于来源正常对照iPSC的星形胶质细胞,来源于患者iPSC的星形胶质细胞表达更多的病理学指标钙结合蛋白S100B和淀粉样肽前提蛋白(APP)。另外,患者来源的星形胶质细胞内活性氧物质的水平也较正常对照组明显增高。在神经分化过程中,患者iPSC与对照组iPSC相比,两者在神经元及星形胶质细胞的分化效率方面差别并不显著。而运用不同来源的星形胶质细胞条件培养基与的患者iPSC神经干细胞共培养后,则发现神经干细胞向神经元和星形胶质细胞的比例明显不同。研究结果表明,在患者iPSC来源的星形胶质细胞的条件培养基中,神经干细胞分化为星形胶质细胞的比例明显增多,分化为神经元的比例明显减少。另外,在共培养体系中,生长在患者iPSC来源星形胶质细胞上的神经元的神经突起较生长在正常iPSC来源星型胶质细胞上的神经元突起短。随着人们越来越重视对星形胶质细胞功能障碍在神经系统疾病中重要作用,本研究认不同类型细胞(神经元及星形胶质细胞)间的相互联系,相互作用对唐氏综合症的发病起重要的作用,因为21号染色体上基因的过度表达而导致的基因剂量效应不仅仅局限在神经元上。
[Abstract]:The production and development of induced pluripotent stem cells (induced Pluripotent Stem Cell or iPSC) provides a new method for the establishment of in vitro cell models of nervous system diseases and for the study of individual specific disease mechanisms. However, most previous studies on neuropathic models were mainly confined to neuronal lesions, often ignoring the role of glial cells in disease phenotypes. In order to simulate the cellular and molecular defects in the pathogenesis of Down syndrome, we reprogrammed human skin fibroblasts to induce pluripotent stem cells by gene transfection. Two of the cell lines were from patients with Down syndrome and the other two were from normal controls. After in vitro culture, the induced pluripotent stem cells were differentiated into neural stem cells, neurons and astrocytes, and the disease model of Down's syndrome was established. The results showed that astrocytes from patients with iPSC expressed more calcium-binding protein S100B and amyloid precursor protein (APP). Than astrocytes derived from normal control iPSC. In addition, the level of reactive oxygen species in astrocytes from patients was significantly higher than that of normal controls. There was no significant difference in the differentiation efficiency of neurons and astrocytes between patients with iPSC and control group with iPSC during neural differentiation. However, the proportion of neural stem cells to neurons and astrocytes was significantly different after co-culture of iPSC neural stem cells with different astrocyte conditioned medium. The results showed that the proportion of neural stem cells differentiated into astrocytes increased and the proportion of neurons decreased significantly in the conditioned medium of astrocytes derived from patients with iPSC. In addition, in the co-culture system, the neurites of the neurons growing on the astrocytes derived from iPSC were shorter than those on the astrocytes derived from normal iPSC. As more and more attention has been paid to the important role of astrocytic dysfunction in nervous system diseases, this study recognizes the interrelationship between different types of cells (neurons and astrocytes). Interaction plays an important role in the pathogenesis of Down's syndrome, because the gene dose effect caused by over-expression of gene on chromosome 21 is not limited to neurons.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

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