Hes1和Mash1基因在脊髓源神经干细胞诱导分化为胆碱能神经元过程中表达的研究

发布时间：2018-01-08 12:00

本文关键词：Hes1和Mash1基因在脊髓源神经干细胞诱导分化为胆碱能神经元过程中表达的研究　出处：《山西医科大学》2009年硕士论文　论文类型：学位论文

【摘要】： 研究背景:神经干细胞作为一种具有自我更新、增殖和可以定向分化为神经元、星型胶质细胞和少突胶质细胞能力的细胞,不仅打破了中枢神经系统发育成熟后不可再生的理论,还为神经系统损伤修复和退行性病变的治疗提供了新的思路[1]。对于神经干细胞的研究,是以保持体外培养的神经干细胞具有活跃的增殖能力为基础的。同时,中枢神经系统各种不同的疾病大多是由于表型和功能不同的神经元病变所致,然而许多研究表明如果将神经干细胞直接移植于受损区,则分化成所需的特异性神经元将很少,难以发挥功能效应。神经干细胞的分化是一个复杂的多因素参与的过程,对于胆碱能神经元的体外分化条件和机制的研究目前尚未达成共识。目的:本部分的研究旨在通过对神经干细胞的分离培养,观察研究其基本生物学特性,了解其增殖分化和体外培养获得大量扩增的条件,为进一步研究神经干细胞的定向分化奠定基础。在体外环境下诱导神经干细胞向胆碱能神经元分化,探讨神经干细胞的体外分化条件及诱导分化机制。方法:将孕龄17 d的胚胎大鼠脊髓组织制备成单细胞悬液,接种到限定性无血清培养液中培养,待形成原代克隆球后传代扩增。对第3代的神经干细胞球进行神经上皮干细胞蛋白(Nestin)免疫荧光检测。然后,将神经干细胞克隆球接种到内置多聚赖氨酸包被的盖玻片的6孔培养板中,应用诱导因子RA、SHH、NT-3和BDNF对神经干细胞进行诱导分化。在37℃、5%CO2饱和湿度的培养箱中分化两周后,用免疫荧光检测胆碱能神经元特异性标志物胆碱乙酰转移酶(ChAT)的表达。结果:获得了大量能自我增殖,表达Nestin,并可在体外长期维持稳定状态的神经干细胞。神经干细胞经诱导因子诱导1 w后,可见分化的细胞呈典型的神经元样细胞形态:胞体饱满而不规则,有多个细长突起;诱导2 w后,神经细胞之间形成了网络状结构,免疫荧光检测,ChAT阳性率达20%。结论:从大鼠胚胎脊髓组织中分离得到的细胞具有不断增殖和自我更新能力,并具有分化为神经元的潜能。SHH、RA、NT-3和BDNF的共诱导可以诱导脊髓源性神经干细胞在体外分化为胆碱能神经元。研究背景:神经干细胞的生长和发育过程的调控涉及多个方面,包括神经细胞特性的决定、细胞数量的控制,细胞分化的格局化和空间控制等。众多基因家族参与了该过程,其中bHLH家族的Hes1和Mash1基因,作为神经发育的相关基因,广泛表达于发育中的中枢和外周神经系统。Hes基因属于负调控型的bHLH转录因子,在维持神经干细胞增殖,使大脑细胞具有正确的数目、形态和细胞排列等方面具有重要的作用。正调控型转录因子Mash1基因,则有促进神经前体细胞向神经元分化作用。正调控型和负调控型bHLH转录因子之间的彼此调控,共同调控着神经干细胞的分化并且决定着其分化方向。本实验通过体外诱导神经干细胞向胆碱能神经元分化,观察Hes1和Mash1基因的表达变化,了解它们在神经干细胞定向分化为胆碱能神经元过程中发挥的作用。目的:通过检测胚胎大鼠脊髓源性神经干细胞定向诱导分化为胆碱能神经元过程中Hes1和Mash1基因的表达变化,探讨神经干细胞的定向分化机制。方法:取体外培养的第三代的神经干细胞,接种于内置多聚赖氨酸包被过的盖玻片的6孔培养板中,通过在培养基中添加诱导因子RA、SHH、NT-3和BDNF,诱导其向胆碱能神经元分化。应用实时定量PCR技术分析神经干细胞及向胆碱能神经元诱导分化1 w和2 w时, Hes1、Mash1和Nestin基因的表达变化。结果:Hes1、Mash1和Nestin mRNA在神经干细胞分化前后表达有变化,诱导1 w时各基因均较对照组表达下降,诱导2 w时表达较诱导1 w时增高,但低于对照组。结论:Hes1、Mash1和Nestin参与了神经干细胞向胆碱能神经元的分化过程,并且发挥了重要的作用。
[Abstract]:Background: neural stem cells as a self-renewal, proliferation and can differentiate into neurons, astrocytes and oligodendrocytes ability of the cells, not only broke the theory of non renewable development of central nervous system after maturity, provides a new way for [1]. neural stem cell research for nervous system damage and repair degenerative disease treatment, is to maintain the in vitro cultured neural stem cells with active proliferation as the foundation. At the same time, the central nervous system is mostly due to various diseases caused by neuron disease phenotype and function of different, but many studies show that if the neural stem cells transplanted directly in the damaged area. It differentiated into specific neurons required is small, it is difficult to play. The functional effect of neural stem cell differentiation is a complex process involved in many factors, for bravery The study of the conditions and mechanisms of the differentiation of alkaline energy neurons in vitro has not yet reached a consensus.
Objective: To study this part through separation of neural stem cell culture, observation and Study on its biological characteristics, understand the proliferation and differentiation of cultured in vitro in the amplification conditions, lay the foundation for further studying on the differentiation of neural stem cells in inducing neural stem cells differentiating into cholinergic neurons in vitro under the environment of in vitro differentiation of neural stem cells and differentiation conditions.
Methods: the gestational age of 17 D of embryonic spinal cord tissue of rats were prepared into single cell suspension was inoculated into the culture medium defined serum-free culture, to form the primary cloning ball passage amplification. The third generation of neural stem cells to neural stem cell (Nestin) immunofluorescence detection. Then, the neurospheres were inoculated into the 6 Hole built-in poly lysine coated coverslip culture plate, induced by factor RA, SHH, NT-3 and BDNF to induce the differentiation of neural stem cells. At 37 degrees, 5%CO2 saturated humidity incubator in two weeks after differentiation, with immune fluorescence detection of cholinergic neuron specific markers of choline acetyltransferase (ChAT) expression.
Results: to obtain a large number of self proliferation, Nestin expression, and in vitro to maintain long-term stable state of neural stem cells. Neural stem cells by inducing differentiation after 1 W visible cells showed the typical neuron like cell morphology: cell body plump and irregular, a plurality of elongated protrusions; induced by 2 W after the network structure formed between nerve cells, immunofluorescence, ChAT positive rate was 20%.
Conclusion: the cells isolated from rat embryonic spinal cord have the ability of continuous proliferation and self-renewal, and have the potential to differentiate into neurons..SHH, RA, NT-3 and BDNF co induction can induce spinal cord derived neural stem cells to differentiate into cholinergic neurons in vitro.
Background: regulation of neural stem cell growth and development process involves many aspects, including the characteristics of neural cells decision, control cell number, cell differentiation pattern and space control. Many gene family is involved in the process, the bHLH family of Hes1 and Mash1 genes, as neural development related genes. The bHLH transcription factors are expressed in the development of the central and peripheral nervous system.Hes gene belongs to the type of negative regulation, in maintaining the proliferation of neural stem cells, brain cells have the correct number, shape and arrangement of cells and other aspects have important role. Positive regulation type transcription factor Mash1 gene can promote neural precursor the role of cells to differentiate into neurons. Positive and negative regulation regulation each other between regulatory bHLH transcription factors, CO regulated the differentiation of neural stem cells and their differentiation determines the direction through this experiment. The differentiation of neural stem cells into cholinergic neurons was induced in vitro. The expression of Hes1 and Mash1 genes were observed, and their roles in the differentiation of neural stem cells into cholinergic neurons were observed.
Objective: through the spinal cord derived neural stem cells of embryonic rat detection differentiated into cholinergic Mash1 neurons and expression of Hes1 gene in the process, to explore the differentiation of neural stem cells. Methods: in vitro cultured neural stem cells of the third generation, the 6 hole with built-in poly-L-lysine the coverslip culture plate, the medium added RA SHH NT-3, inducing factor, and BDNF induced neuronal differentiation into cholinergic. Analysis of neural stem cells and differentiation to cholinergic neurons induced by 1 W and 2 W, Hes1 using real-time PCR technique. The expression of Mash1 and Nestin gene results: Hes1, Mash1 and Nestin in mRNA cell differentiation and expression changes of neural stem induced by 1 W genotypes were higher than those in control group decreased 2 W induced increased expression is induced by 1 W, but lower than the control group.
Conclusion: Hes1, Mash1 and Nestin participate in the differentiation process of neural stem cells to cholinergic neurons and play an important role.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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