关于CD133作为成纤维细胞转分化为神经干细胞筛选标记的研究

发布时间：2018-03-23 02:34

本文选题：神经干细胞　切入点：转分化　出处：《昆明理工大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景与目的:神经干细胞(neural stem cells,NSCs)指具有分化为神经元、星形胶质细胞及少突胶质细胞的能力,能自我更新并足以提供大量脑组织细胞的细胞群。因神经干细胞具备自我更新和多分化潜能的两个基本特性,以及迁移功能、低免疫性和良好的组织融合性的优点,故而成为细胞移植治疗神经系统疾病良好的移植材料,但是如何体外获得并分选出高纯度的神经干细胞便成为其推向临床治疗关键。在前期的实验中,我们发现:在成纤维细胞在转分化为神经干细胞的过程中,CD133这一细胞表面膜蛋白的表达量在前5天呈现出显著升高的趋势,其后维持高表达状态。所以本课题旨在确定CD133这一蛋白能否作为成纤维细胞转分化为神经干细胞过程中的筛选标记,从而为体外分选高纯度神经干细胞提供理论依据,也为进一步研究转分化机制及神经干细胞应用奠定良好的基础。材料与方法:取65天的猕猴耳部组织获得成纤维细胞,在35mm的小皿中用包含Sox2、Oct4和Klf4三个转录因子的逆病毒感染3×104个新生猴成纤维细胞两次。第三天用胰酶消化,往铺过5 mg/mLlaminin的一个六孔板中接入l× 105个细胞,并加入3 mL诱导培养基。在诱导第5天和第七天,分别去掉Y27632和丙戊酸,在第11天时换为神经干细胞培养基。期间,对每一天的细胞进行CD133的免疫荧光染色,观察CD133的表达情况。在第七天时,对细胞进行CD133流式分选,分别收集阳性细胞和阴性细胞继续培养,并进行神经鉴定。结果:(1)用Sox2、Oct4和Klf4的逆转录病毒感染成纤维细胞后,每一种病毒的感染效率基本都达到了 80%。(2)虽然随着天数的增加,由于细胞大量死亡,CD133的表达有一定增强,但其表达量在宏观上并没有显著增多。(3)经鉴定,在转分化第7天利用流式细胞术分选出的CD133阳性细胞群可以成功转分化为神经干细胞,而CD133阴性细胞不能转分化为神经干细胞。结论:CD133可以作为成纤维细胞转分化为神经干细胞过程中的筛选标记。
[Abstract]:Background & AIM: neural stem cells have the ability to differentiate into neurons, astrocytes and oligodendrocytes. A group of cells capable of self-renewal and sufficient to provide a large number of brain tissue cells. Because neural stem cells have two basic characteristics of self-renewal and multi-differentiation potential, as well as the advantages of migration, low immunity and good tissue fusion, So it has become a good transplantation material for the treatment of nervous system diseases, but how to obtain and select high purity neural stem cells in vitro is the key to clinical treatment. We found that the expression of CD133, a membrane protein on the surface of fibroblasts, increased significantly in the first five days during the process of transforming fibroblasts into neural stem cells. The aim of this study was to determine whether the CD133 protein can be used as a screening marker in the process of fibroblast transdifferentiation into neural stem cells, so as to provide a theoretical basis for the separation of high purity neural stem cells in vitro. It also laid a good foundation for further study on the mechanism of transdifferentiation and the application of neural stem cells. Materials and methods: the fibroblasts were obtained from the ear tissue of rhesus monkey after 65 days. The 3 脳 104 neonatal monkey fibroblasts were infected twice with antivirus containing three transcription factors of Sox2 Oct4 and Klf4 in a small dish of 35mm. On the third day, the cells were digested with trypsin, and 1 脳 105 cells were injected into a six-well plate spread over 5 mg/mLlaminin. On the 5th and 7th day of induction, Y27632 and valproic acid were removed and replaced into neural stem cell culture medium on the 11th day. During the induction, the cells were stained with CD133 immunofluorescence staining on each day. The expression of CD133 was observed. On the seventh day, CD133 flow sorting was carried out on the cells, positive and negative cells were collected and cultured, and the nerve was identified. Results: the fibroblasts were infected with the retrovirus of Sox2Oct4 and Klf4. The infection efficiency of each virus basically reached 80%.) although the expression of CD133 increased with the increase of days, the expression of CD133 was not significantly increased in macroscopically. On the 7th day of transdifferentiation, CD133 positive cells were successfully transformed into neural stem cells by flow cytometry. CD133 negative cells could not be transformed into neural stem cells. Conclusion: CD133 can be used as a screening marker in the process of fibroblast transdifferentiation into neural stem cells.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q42

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