无血清体系直接高效诱导大鼠ES细胞分化为神经细胞的研究

发布时间：2018-04-09 17:01

本文选题：大鼠胚胎干细胞　切入点：神经分化　出处：《浙江理工大学》2010年硕士论文

【摘要】：神经退行性疾病(Neurodegenerative disease)是一类慢性、进行性神经系统疾病,近些年来,随着社会的老龄化程度加剧,神经退行性疾病在所有疾病中占有越来越重要的地位。传统的观念认为,神经细胞是没有再生能力的,所以之前的研究基本上都集中在改善神经系统疾病的受损症状和防止疾病损害加强的治疗方法上。上个世纪90年代,随着神经干细胞的发现人们改变了这种观念,但是关于神经系统损伤疾病仍然没有有效的治疗方法,因此中枢神经系统损伤修复是人类面临的巨大挑战。随着胚胎干细胞的分离获得和成功建系,人们开始将胚胎干细胞用于细胞替代治疗的研究。胚胎干细胞是一种全能性干细胞,其向神经方向诱导分化成为神经细胞移植的一种广泛的细胞来源,如果移植的神经细胞可正常发挥功能,这将会从根本上治疗一些神经系统疾病。现在关于胚胎干细胞分化为神经细胞的方法主要有形成拟胚体、基质细胞共培养、细胞因子诱导培养法和遗传修饰法等方法,但大都存在一些诸如需要基于细胞滋养的培养过去麻烦、分化效率太低或存在安全性等问题。胚胎干细胞分化为神经细胞做为细胞替代治疗需要研究一种简单而有效的分化方法。本论文采用无血清培养基,首先用特定的分化培养液可以高效地将大鼠胚胎干细胞诱导分化为神经球干细胞,免疫荧光和RT-PCR的实验结果表明,这些细胞可以表达神经干细胞相关的标记,然后通过添加不同的分化细胞生长因子可以将胚胎干细胞定向地诱导分化为为运动能神经元和多巴胺能神经元,这表明我们诱导的神经干细胞具有良好的可塑性,可以分化为不同的神经细胞。在体内实验中,我们首先用携带红色荧光蛋白H794的慢病毒感染神经球干细胞,筛选纯化后将表达红色荧光蛋白的神经球干细胞进行初步分化,然后移植注射到成年大鼠脑黑质纹状体内,观察存活及分化情况。注射20d后,取实验大鼠进行全身固定,取脑组织然后制作冰冻切片,免疫荧光染色后在荧光显微镜下可以观察到注射的红色荧光蛋白感染的细胞。本实验建立了一种简单、快速、高效的胚胎干细胞分化为神经干细胞的方法,并进行进一步分化为特定神经细胞,初步尝试进行了体内实验,胚胎干细胞诱导分化来的神经细胞成了神经系统疾病重要的细胞来源。通过本实验,我们得出在无血清条件下,通过单层细胞粘附培养可以获得神经干细胞,本实验为神经干细胞用于神经系统性疾病的治疗打下了基础。
[Abstract]:Neurodegenerative disease is a kind of chronic and progressive neurological disease. In recent years, with the aggravation of society, neurodegenerative diseases play a more and more important role in all diseases.The conventional view is that nerve cells are not regenerative, so previous studies have focused on improving the symptoms of neurological diseases and strengthening treatments to prevent them.In the 1990s, with the discovery of neural stem cells, people changed this idea, but there is still no effective treatment for nervous system injury diseases, so the repair of central nervous system injury is a great challenge to human beings.With the isolation and successful establishment of embryonic stem cells, embryonic stem cells have been used in cell replacement therapy.Embryonic stem cells are omnipotent stem cells, which are induced to differentiate into a wide range of cell sources for neural transplantation, if the transplanted nerve cells function normally.This will fundamentally cure some neurological diseases.At present, the methods of differentiation of embryonic stem cells into nerve cells mainly include the formation of embryoid body, co-culture of stromal cells, cytokine induction culture and genetic modification, etc.However, most of them have some problems, such as the past troubles of cell-based culture, low differentiation efficiency or safety.The differentiation of embryonic stem cells into neural cells requires a simple and effective method of differentiation.In this paper, serum-free medium was used to induce the differentiation of rat embryonic stem cells into neuronal stem cells. The results of immunofluorescence and RT-PCR showed that rat embryonic stem cells could be induced to differentiate into neural globular stem cells.These cells can express markers associated with neural stem cells, which can then be induced to differentiate into motor and dopaminergic neurons by adding different differentiated cell growth factors.This indicates that the neural stem cells we induce have good plasticity and can differentiate into different nerve cells.In vivo experiments, we first infected neural ball stem cells with lentivirus carrying red fluorescent protein H794. After screening and purification, the neural ball stem cells expressing red fluorescent protein were preliminarily differentiated.Then transplanted into the substantia nigra striatum of adult rats to observe the survival and differentiation.After 20 days of injection, the experimental rats were fixed in the whole body, and then frozen sections were made from the brain tissue. After immunofluorescence staining, the cells infected with red fluorescent protein could be observed under fluorescence microscope.In this study, a simple, rapid and efficient method for differentiation of embryonic stem cells into neural stem cells was established, and further differentiation into specific nerve cells was carried out.Embryonic stem cells induce differentiation of nerve cells into an important source of nervous system diseases.Through this experiment, we concluded that neural stem cells could be obtained by monolayer cell adhesion culture in serum-free condition, which laid a foundation for the treatment of nervous system diseases by neural stem cells.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329;R741

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