干细胞体外诱导为类毛细胞的实验研究

发布时间：2018-08-05 15:10

【摘要】：第一部分神经干细胞的分离培养、鉴定及标记目的神经干细胞(NSC)的研究已逐渐成为国内、外基础及临床医学各专业的重要研究课题。NSC是一种成体干细胞，存在于胚胎和成年哺乳动物中枢神经系统的多个区域，属多能干细胞，，在一定的微环境中具有自我更新和向神经元、星形胶质细胞、少突胶质细胞分化的能力，且具有极强的可塑性，利用NSC进行细胞治疗或基因治疗有很好的临床应用前景。豚鼠在耳科学基础研究中具有独特的优势，并得到广大耳科学研究者的广泛应用，建立一种简便、可行的豚鼠NSC培养方法将为NSC在耳科学领域研究的深入开展提供细胞支持，但目前关于豚鼠NSC的培养方法介绍的并不多。本实验旨在建立一种较为简便、可靠的豚鼠NSC分离、培养、标记方法，为耳科学领域NSC的进一步深入研究提供帮助。材料与方法分离新生豚鼠海马组织进行NSC的原代、传代培养及诱导分化培养，倒置显微镜下观察培养细胞的形态特征，培养的细胞行Nestin免疫细胞化学，Western Biot检测，对诱导细胞行NSE、GFAP免疫荧光化学观察，以鉴定所培养的细胞为NSC；用荧光染料DAPI标记干细胞，计算原代及传代标记率。结果 1．原代培养见细胞呈大小不一、悬浮生长的细胞团，边界清晰，细胞团中细胞排列较为致密。光镜下传代培养的细胞形态特点与原代培养无明显差异。2．原代及传代培养的细胞团Nestin阳性表达，诱导分化后的细胞NSE、GFAP阳性表达。3．经荧光染料DAPI标记后，标记成功率可达93.4％，经传代8次后荧
[Abstract]:The first part is the isolation, culture, identification and labeling of neural stem cells (NSC). NSC is a kind of adult stem cells, which exist in many regions of the central nervous system of embryonic and adult mammals and belong to pluripotent stem cells. In a certain microenvironment, it has the ability of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes, and has a strong plasticity. The use of NSC for cell therapy or gene therapy has a good clinical application prospects. Guinea pigs have unique advantages in the basic research of ear science and have been widely used by many ear researchers. The establishment of a simple and feasible method of guinea pig NSC culture will provide cell support for the further research of NSC in the field of ear science. But at present, the methods of guinea pig NSC culture are not much introduced. The aim of this study was to establish a simple and reliable method for the isolation, culture and labeling of guinea pig NSC, which would be helpful for the further study of NSC in the field of ear science. Materials and methods the hippocampal tissues of newborn guinea pigs were isolated and cultured with NSC. The morphological characteristics of cultured cells were observed under inverted microscope. The cultured cells were detected by Nestin immunocytochemistry and Western Biot. The induced cells were observed by immunofluorescence staining to identify the cultured cells as NSC.The labeled stem cells were labeled with fluorescent dye DAPI and the primary and passage labeling rates were calculated. Results 1. The size of cells was different in primary culture. The boundary of cell clusters was clear and the cells in the cell clusters were compact. 2. The morphological characteristics of the cells cultured under light microscope were not significantly different from those of the primary culture. 2. The positive expression of Nestin was observed in primary and subcultured cells, and the positive expression of NSE-GFAP was induced in differentiated cells. The cells were labeled with fluorescent dye DAPI. The success rate of labeling was 93.44.After passage for 8 times, it was fluorescent.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R329

【相似文献】