Notch途径在中枢神经系统发育过程中的作用
发布时间：2018-08-27 06:26
【摘要】：干细胞一直吸引着人们的研究,是因为它可以在维持自身未分化状态(自我更新)的同时产生多种子代细胞。神经干细胞(NSC)是神经系统发育的基础,它们为数不多却具有高度的可塑性,能够增殖分裂、获得位置信息、继而分化出不同的子代细胞(神经元和胶质细胞),最终发育成为整个神经系统。NSC的这些基本特征使其成为治疗多种神经退行性疾患和中枢神经损伤的最有前途的侯选方法,然而,在充分理解NSC的增殖和分化调控机理之前,将NSC应用于临床治疗是不可能的。因此研究NSC的增殖和分化机理不仅有助于神经发育的理论探讨,更是将NSC应用于临床的前提。 目前已知NSC的增殖分化受到多种细胞自主性和细胞非自主性因素的调节,其中以细胞膜受体Notch为基础的信号途径在神经干细胞增殖分化的调控中发挥重要作用。Notch途径对于神经发生的各个方面均有重要的调控作用,如维持NSC的未分化状态,调控NSC分化过程中的命运选择,以及调控子代细胞的最终成熟。但是关于Notch途径发挥调控作用的具体机制,即它是如何调控NSC和神经祖细胞(INP)的增殖和分化,尤其是体内的状况,还有待于进一步的研究。 在本课题中,我们拟构建中枢神经系统(CNS)特异性阻断Notch途径的转基因小鼠,并在此基础上在体观察Notch途径阻断后NSC的各种异常表型,包括NSC自身的增殖分化的异常、Notch阻断的NSC分化出的子代细胞是否有异常等。最终,我们希望能够在体阐明Notch途径在CNS发育的各个过程中所发挥的不同作用,并初步探讨其作用的机制。 本课题所取得的成果分述如下: 1、首先,我们成功构建了NesCre转基因小鼠,并鉴定出其Cre重组酶的表达范围主要包括基底前脑和中脑腹侧等。再通过NesCre小鼠和先期构建成功的RBP-J-floxed小鼠交配,获得在CNS特异性剔除Notch途径中的关键转录因子RBP-J的条件性基因剔除小鼠。 2、其次,在剔除小鼠中,我们首先观察到NSC分化的异常。在胚胎发育的早期(即E11.5时)RBP-J剔除区域的组织体外培养神经球的数目有所增多。但是,这些增多的神经球却以神经元祖细胞(INP)为主,提示RBP-J剔除后存在NSC向INP的提前分化。在胚胎发育的晚期(E17.5和新生时期),由于NSC池的提前消耗,RBP-J剔除区域的体外培养神经球数目降低,在体观察NSC数目也减少,同时伴随有神经元和胶质细胞等子代细胞的分化增加。除此以外,在E11.5时我们观察到神经元的发生却有所降低。这些结果提示在正常胚胎发育过程中RBP-J介导的Notch途径可以抑制NSC向INP的分化,并有可能对某些早期生成的神经元的发生具有重要的调控作用。 3、再次,由RBP-J剔除的NSC分化而来的神经元的形态也有所改变。NSC中的Notch途径阻断后,不仅向神经元的分化增加,而且分化出来的神经元的突起分支数也增多。 4、最后,我们进行了深入的调控机制研究。芯片结果提示一些miRNA分子在NSC增殖分化过程中可能发挥重要的调控作用。而在神经元形态发育方面,我们发现microRNA342-5p可以促进神经元的突起分支长出,而Notch途径可以抑制microRNA342-5p的表达,从而实现对神经元形态发育的调控作用。 综上所述,我们的研究阐明了经典Notch途径在NSC向INP分化过程中的作用,提示microRNA分子可能参与其中;Notch途径在神经元的终末成熟过程中也发挥着重要的作用,microRNA342-5p参与其中,受到Notch途径的调控。这些研究为我们进一步理解CNS发育提供了详实的实验数据,尤其是Notch途径与microRNA分子的相互作用研究,为Notch途径调控CNS发育的机制研究提供了新的理论知识。这些研究也为将来Notch途径作为临床治疗潜在靶点提供了坚实的理论基础。
[Abstract]:Stem cells have attracted much attention because they can produce a variety of offspring while maintaining their undifferentiated state (self-renewal). Neural stem cells (NSCs) are the basis for the development of the nervous system. They are small but highly plastic, capable of proliferating and dividing, obtaining location information, and then differentiating into different molecules. These basic characteristics of NSC make it the most promising candidate for treatment of many neurodegenerative diseases and central nervous system injury. However, it is impossible to apply NSC to clinical treatment until the mechanism of regulation of proliferation and differentiation of NSC is fully understood. Therefore, the study of the proliferation and differentiation mechanism of NSC is not only conducive to the theoretical study of neural development, but also a prerequisite for the clinical application of NSC.
It is known that the proliferation and differentiation of NSC are regulated by a variety of cellular autonomy and non-autonomic factors. Notch-based signaling pathway plays an important role in the regulation of neural stem cell proliferation and differentiation. Notch pathway plays an important role in all aspects of neurogenesis, such as maintaining the absence of NSC. Differentiation status, fate selection during differentiation of NSC and final maturation of progeny cells are regulated. However, the specific mechanism of Notch pathway, that is, how it regulates the proliferation and differentiation of NSC and neural progenitor cells (INP), especially in vivo, remains to be further studied.
In this study, we intend to construct transgenic mice that specifically block Notch pathway by the central nervous system (CNS), and then observe the abnormal phenotypes of NSC in vivo after Notch pathway blockade, including the abnormal proliferation and differentiation of NSC itself, and whether the offspring cells differentiated from NSC blocked by Notch are abnormal or not. To elucidate the different roles of Notch pathway in the development of CNS in vivo and to explore its mechanism.
The results of this research are as follows:
1. First, we successfully constructed NesCre transgenic mice and identified the expression range of Cre recombinase mainly in the basal forebrain and ventral midbrain. Mice.
2. Secondly, we observed the abnormal differentiation of NSC in the rejected mice. In the early embryonic development (E11.5), the number of cultured neurospheres in the rejected RBP-J region increased in vitro. However, these increased neurospheres were mainly neuron progenitor cells (INP), suggesting that there was an early differentiation of NSC into INP after RBP-J rejection. In the late stages of fetal development (E17.5 and neonatal period), the number of cultured neurons in the RBP-J exclusion region decreased due to the early consumption of NSC pools, and the number of NSCs decreased in vivo, accompanied by an increase in the differentiation of neurons and glial cells. These results suggest that the Notch pathway mediated by RBP-J may inhibit the differentiation of NSC into INP during normal embryonic development, and may play an important role in the regulation of neurogenesis in early stage.
3. Thirdly, the morphology of neurons differentiated from NSC excluded by RBP-J also changed. After the blockade of Notch pathway in NSC, not only the differentiation of neurons increased, but also the number of neurite branches of differentiated neurons increased.
4. Finally, we conducted in-depth study of the regulatory mechanisms. The results of the microarray suggest that some of the microRNAs may play an important role in the proliferation and differentiation of NSC. Thus, it can regulate the morphological development of neurons.
In summary, our study elucidates the role of the classical Notch pathway in the differentiation of NSC into INP, suggesting that microRNA molecules may be involved; Notch pathway also plays an important role in the terminal maturation of neurons, in which microRNA342-5p is involved and regulated by Notch pathway. Development provides detailed experimental data, especially the interaction between Notch pathway and microRNA molecules, and provides new theoretical knowledge for the mechanism of Notch pathway regulating CNS development.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R33
本文编号：2206402
[Abstract]:Stem cells have attracted much attention because they can produce a variety of offspring while maintaining their undifferentiated state (self-renewal). Neural stem cells (NSCs) are the basis for the development of the nervous system. They are small but highly plastic, capable of proliferating and dividing, obtaining location information, and then differentiating into different molecules. These basic characteristics of NSC make it the most promising candidate for treatment of many neurodegenerative diseases and central nervous system injury. However, it is impossible to apply NSC to clinical treatment until the mechanism of regulation of proliferation and differentiation of NSC is fully understood. Therefore, the study of the proliferation and differentiation mechanism of NSC is not only conducive to the theoretical study of neural development, but also a prerequisite for the clinical application of NSC.
It is known that the proliferation and differentiation of NSC are regulated by a variety of cellular autonomy and non-autonomic factors. Notch-based signaling pathway plays an important role in the regulation of neural stem cell proliferation and differentiation. Notch pathway plays an important role in all aspects of neurogenesis, such as maintaining the absence of NSC. Differentiation status, fate selection during differentiation of NSC and final maturation of progeny cells are regulated. However, the specific mechanism of Notch pathway, that is, how it regulates the proliferation and differentiation of NSC and neural progenitor cells (INP), especially in vivo, remains to be further studied.
In this study, we intend to construct transgenic mice that specifically block Notch pathway by the central nervous system (CNS), and then observe the abnormal phenotypes of NSC in vivo after Notch pathway blockade, including the abnormal proliferation and differentiation of NSC itself, and whether the offspring cells differentiated from NSC blocked by Notch are abnormal or not. To elucidate the different roles of Notch pathway in the development of CNS in vivo and to explore its mechanism.
The results of this research are as follows:
1. First, we successfully constructed NesCre transgenic mice and identified the expression range of Cre recombinase mainly in the basal forebrain and ventral midbrain. Mice.
2. Secondly, we observed the abnormal differentiation of NSC in the rejected mice. In the early embryonic development (E11.5), the number of cultured neurospheres in the rejected RBP-J region increased in vitro. However, these increased neurospheres were mainly neuron progenitor cells (INP), suggesting that there was an early differentiation of NSC into INP after RBP-J rejection. In the late stages of fetal development (E17.5 and neonatal period), the number of cultured neurons in the RBP-J exclusion region decreased due to the early consumption of NSC pools, and the number of NSCs decreased in vivo, accompanied by an increase in the differentiation of neurons and glial cells. These results suggest that the Notch pathway mediated by RBP-J may inhibit the differentiation of NSC into INP during normal embryonic development, and may play an important role in the regulation of neurogenesis in early stage.
3. Thirdly, the morphology of neurons differentiated from NSC excluded by RBP-J also changed. After the blockade of Notch pathway in NSC, not only the differentiation of neurons increased, but also the number of neurite branches of differentiated neurons increased.
4. Finally, we conducted in-depth study of the regulatory mechanisms. The results of the microarray suggest that some of the microRNAs may play an important role in the proliferation and differentiation of NSC. Thus, it can regulate the morphological development of neurons.
In summary, our study elucidates the role of the classical Notch pathway in the differentiation of NSC into INP, suggesting that microRNA molecules may be involved; Notch pathway also plays an important role in the terminal maturation of neurons, in which microRNA342-5p is involved and regulated by Notch pathway. Development provides detailed experimental data, especially the interaction between Notch pathway and microRNA molecules, and provides new theoretical knowledge for the mechanism of Notch pathway regulating CNS development.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R33
【引证文献】
相关硕士学位论文 前1条
1 王俊伟;ADAM10在大脑皮层神经细胞发育过程中调节作用的研究[D];浙江大学;2012年
，本文编号：2206402
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2206402.html
最近更新
教材专著
