Notch信号通路在神经干细胞分化过程中的调节作用研究

发布时间：2018-06-07 01:24

本文选题：神经管畸形 + 神经干细胞　；参考：《山西医科大学》2009年硕士论文

【摘要】： 神经系统是机体最重要和最复杂的系统,起源于神经管(neuarl tube)和神经峭,神经管形成中枢神经系统(central nevrous system, CNS)(脑和脊髓),神经峭形成周围神经系统的神经节等。CNS正常发生的关键是神经板内外相关组织细胞行为的准确进行和协同,神经管的形成是该过程结束的标志。神经管发生是一个重要的涉及到建立中枢神经系统原基的胚胎学事件,是指从神经板出现到神经管关闭的发育过程。神经管管壁最初是由一层较厚的假复层上皮组成,称为神经上皮(neuroepithelium)。神经上皮不断增殖的同时细胞也逐渐开始进行迁移和分化,逐渐形成三层结构的管壁,由内向外依次为室管膜层,套层和边缘层。在此过程中,神经上皮细胞处于活跃的细胞增殖周期中,为具有多种分化潜能的神经干细胞(neutal stem cell, NSC),伴随着分化演变。神经上皮中神经干细胞的增殖迁移以及分化是神经系统发育的关键环节。研究发现神经管形成时期极易受多种内外因素的干扰而致畸,神经管缺陷(neural tube defects, NTDs)就是其中发病率最高的一种,表现为各种脑和脊髓的发育畸形。多年来的研究提示NTDs的发生与神经上皮的异常发育密切相关。从基因水平而言,神经上皮的发育过程是一系列基因按照高度特异的时空模式表达并相互作用的结果,但迄今对此复杂过程的基因表达与调控的了解还很少。在各种调控机制中,Notch信号通路(Notch signaling pathway)与神经管的发育有着密切关系,并且由它介导的“旁侧抑制(lateral inhibition)”机制被认为是决定NSC分化命运的一个关键环节。反式维甲酸(all-trans retinoic acid, ATRA)是维生素A在体内的正常代谢产物,它是一种作用很强的诱导分化剂,在神经管的形成过程中,它具有很重要的作用。由维甲酸诱导形成的NTDs模型在科学研究中已经得到了广泛的应用,但其具体的致畸作用机制尚未见文献报道。鉴于NSC增殖与分化的机制在神经管形成时期具有重要意义,所以本课题利用Western blot、Real-time PCR、双荧光素酶报告基因系统、神经干细胞体外培养、质粒转染及免疫荧光染色等技术,初步研究了ATRA对NSC分化过程中Notch信号通路的具体调节途径,为进一步探讨Notch信号通路在胚胎神经系统发育中的作用机制,尤其是对神经上皮细胞的增殖分化的影响以及在神经管发育和NTDs中可能分子机制奠定了理论基础和实验依据。第一章神经干细胞分化过程中Notch信号通路相关基因的表达变化目的探讨神经干细胞体外分离培养的方法、增殖特点和多向分化的特性。使用ATRA作用于处于分化状态的NSCs,检测其对Notch信号通路相关基因(Notch 1, Musashi 1, Numb, Presenilin 1, Rbpj, Hes 1, Sox 1, Mash 1和Neurogenin 2)表达变化的影响。方法取15天C57BL/6胎鼠的大脑皮质组织,采用机械分离的方法获得神经干细胞,并应用无血清神经干细胞培养基进行原代和传代培养。对培养的神经干细胞进行Nestin(巢蛋白)免疫细胞化学鉴定;将获得的神经干细胞进行体外分化,对分化结果进行NF(Neurofilament,神经丝蛋白,为神经元标志抗原)、GFAP(Glial Fibrillary Acidic protein,胶质原纤维酸性蛋白,为星形胶质细胞标志抗原)和GALC(Galactocerebroside,半乳糖脑苷脂,为少突胶质细胞标志抗原)免疫细胞化学鉴定。并在神经干细胞增殖和分化过程中进行细胞形态学观察。将第5代经鉴定过的神经干细胞分为两组:对照组使用分化培养基进行培养,实验组就是在对照组的基础上再同时加入1μmol/L ATRA。每组分别取NSC(分化0d),分化1,3,5,7d共5个时间点进行实验,每个时间点分别重复三次。在各个时间点通过实时荧光定量PCR反应检测Notch通路相关基因在mRNA表达水平的相对变化情况,通过Western Blot测定Notch1蛋白胞内段NICD的相对表达变化。结果采用机械分离和无血清培养的方法可以获得大量的神经干细胞,对其进行Nestin免疫细胞化学鉴定为阳性,证明为神经干细胞;神经干细胞体外分化可获得各种神经终末细胞,NF、GFAP和GALC鉴定结果均为阳性,证明可分化为神经元、星形胶质细胞和少突胶质细胞。使用ATRA后,Notch 1表达降低,其胞内段NICD的生成量也降低,Musashi 1表达增高,Numb表达先增高后降低,Presenilin 1表达增高,Rbpj表达先降低后增高,Hes 1表达降低,Sox 1表达降低,Mash 1表达降低,Neurogenin 2表达降低。结论应用机械分离和无血清神经干细胞培养基培养的方法可获得大量的神经干细胞,这些神经干细胞具有多向分化的潜能,可分化为神经元等多种神经终末细胞。 Notch 1、Hes 1表达的降低和NICD生成量的降低说明ATRA可能直接或间接地通过抑制Notch 1的表达从而促进了神经干细胞的分化,Musashi 1表达增高以及Numb在后期表达相应的降低可能与Notch 1胞内段NICD减少从而产生一种反馈性调节机制有关,Presenilin 1表达增高以及Rbpj表达在后期的增高也可能属于这种反馈调节机制。Sox 1表达的降低可能并不是因为ATRA的作用引起的,而是因为Hes 1表达降低产生反馈调节引起的。Mash 1和Neurogenin 2表达降低说明,使用ATRA后NSC向神经元方向的分化减少了。第二章γ-分泌酶在神经干细胞分化过程中的作用目的建立基于Gal4-VP16/UAS系统和双荧光素酶报告基因系统检测神经干细胞分化过程中γ分泌酶活性的方法。对神经干细胞分化过程中γ分泌酶的活性、酶解产物NICD和活性中心—早老素1(Presenilin 1, PS1)进行检测,为后期深入研究神经干细胞分化过程的调节机制提供一个重要的实验基础。检测神经干细胞分化过程中ATRA对γ分泌酶活性的影响。方法将编码携带有转录激活因子Gal4-VP16的小鼠Notch1跨膜与胞内段的质粒Notch1△E-GVP,编码萤火虫荧光素酶基因和上游激活序列(UAS)的质粒MH100,以及海肾荧光素酶质粒pRL-CMV,用脂质体转染法转入处于分化状态的神经干细胞,利用Gal4-VP16/UAS系统和双荧光素酶报告基因系统测定神经干细胞分化过程中γ分泌酶的活性;通过Western blot技术检测γ分泌酶酶解产物NICD的生成量;采用实时荧光定量PCR测定γ分泌酶活性中心PS1的表达。结果在γ分泌酶抑制剂DAPT作用下,γ分泌酶活性呈剂量依赖性降低,NICD的生成量也同步减少,而其催化中心组份PS1的表达则呈反馈性的同步增高。使用ATRA作用于处于分化状态的NSCs后,γ分泌酶的活性增高了。结论利用Gal4-VP16/UAS系统和双荧光素酶报告基因系统来检测神经干细胞分化过程中γ分泌酶的活性,为研究神经干细胞分化过程的调节提供了一个重要的实验方法。在神经干细胞分化过程中,从γ分泌酶对Notch通路的这个调控范围来说,ATRA抑制Notch通路的作用在于抑制Notch 1的表达,而不在于直接抑制γ分泌酶的活性及其催化组份早老素1的表达。
[Abstract]:The nervous system is the most important and complex system of the body, which originates from the Neuarl tube and the nerve, and the nerve canal forms the central nevrous system (CNS) (brain and spinal cord). The key to the normal hair growth of the ganglion, such as the ganglion that forms the peripheral nervous system, is the accuracy of the behavior of the cells inside and outside the nerve plate. The formation of neural tube is the sign of the end of the process. Neural tube formation is an important embryological event involving the establishment of the central nervous system. It refers to the development process from the nerve plate to the closure of the nerve canal. The nerve tube wall was initially composed of a thicker layer of false lamina, called the neuroepithelium (neuro Epithelium). At the same time, the cells gradually proliferate and the cells gradually begin to migrate and differentiate, and gradually form the three layer structure of the tube wall, from the inside to the outer layer, the interlayer and the marginal layer. In this process, the neuroepithelial cells are in the active cell proliferation cycle, and are the neural stem cells with multiple differentiation potential (neutal Stem cell, NSC), with the differentiation and evolution. The proliferation, migration and differentiation of neural stem cells in the neural epithelium are the key link in the development of neural system. It is found that the period of neural tube formation is easily teratogenic by the interference of various internal and external factors. Neural tube defects (neural tube defects, NTDs) is one of the highest incidence of the neural tube (NTDs). Various brain and spinal deformities. Years of research have suggested that the occurrence of NTDs is closely related to the abnormal development of the neuroepithelium. From the level of gene, the development of the neuroepithelium is the result of a series of genes expressed and interacting in a highly specific spatio-temporal pattern, but the gene expression and regulation of this complex process have been present to date. There is little understanding.
In various regulatory mechanisms, the Notch signaling pathway (Notch signaling pathway) is closely related to the development of neural tubes, and the mechanism of "side inhibition (lateral inhibition)" mediated by it is considered to be a key link in determining the fate of NSC differentiation. Trans retinoic acid (all-trans retinoic acid, ATRA) is in the body of vitamin A. Normal metabolites, which are a strong inducing differentiation agent, play an important role in the formation of neural tubes. The NTDs model induced by retinoic acid has been widely used in scientific research, but its specific mechanism for teratogenicity has not been reported in the literature. In view of the mechanism of NSC proliferation and differentiation The period of neural tube formation is of great significance, so this subject uses Western blot, Real-time PCR, double luciferase reporter gene system, neural stem cells in vitro culture, plasmid transfection and immunofluorescence staining and other techniques, preliminary study the specific regulation pathway of ATRA on NSC signaling pathway in the process of NSC differentiation, in order to further explore Notch letter. The mechanism of the signaling pathway in the development of embryonic neural system, especially the effect on the proliferation and differentiation of neuroepithelial cells, and the theoretical basis and experimental basis for the development of neural tube and possible molecular mechanism in NTDs.
Chapter one expression changes of Notch signaling pathway related genes during neural stem cell differentiation
objective
To investigate the methods of isolation and culture of neural stem cells in vitro, the characteristics of proliferation and multidirectional differentiation. The effects of ATRA on the differentiated NSCs were used to detect the changes in the expression of Notch signaling pathway related genes (Notch 1, Musashi 1, Numb, Presenilin 1, Rbpj, Hes 1, Sox 1, Mash 1 and Neurogenin 2).
Method
The neural stem cells were obtained by mechanical separation in the cerebral cortex of C57BL/6 fetal rat for 15 days. The cultured neural stem cell culture medium was used for primary and subculture. The cultured neural stem cells were identified by Nestin (nestin) immunocytochemical identification, and the obtained neural stem cells were differentiated in vitro, and the differentiation results were obtained. NF (Neurofilament, neurofilament protein, neuron marker antigen), GFAP (Glial Fibrillary Acidic protein, glial fibrillary acidic protein, astrocyte marker antigen) and GALC (Galactocerebroside, galactose brain glycoside, oligodendrocyte marker antigen) immunocytochemical identification, and proliferation and proliferation in neural stem cells. Cell morphology was observed during the differentiation process.
The fifth generation of identified neural stem cells were divided into two groups: the control group was cultured with the differentiation medium. The experimental group was added to the control group and added 1 mu mol/L ATRA. at the same time to take NSC (differentiation 0d), and the 1,3,5,7d was divided into three times at each time point. The relative changes in the expression level of the Notch pathway related genes were detected by the fluorescence quantitative PCR reaction, and the relative expression of the NICD in the intracellular segment of the Notch1 protein was measured by Western Blot.
Result
A large number of neural stem cells can be obtained by mechanical separation and serum-free culture. Nestin immunocytochemical identification of them is positive. It is proved to be neural stem cells. The differentiation of neural stem cells in vitro can obtain all kinds of nerve terminal cells. NF, GFAP and GALC identify the positive results, which prove that it can be differentiated into neurons and astrocytes. Cells and oligodendrocytes.
After the use of ATRA, the expression of Notch 1 decreased, the production of NICD in the intracellular segment decreased, the expression of Musashi 1 increased, the expression of Numb increased first and then decreased, the expression of Presenilin 1 increased, the expression of Rbpj decreased first, the expression of Hes 1 decreased, the expression of Sox 1 decreased, the expression of Mash 1 decreased, Neurogenin 2 was reduced.
conclusion
A large number of neural stem cells can be obtained by means of mechanical separation and culture medium culture of serum-free neural stem cells. These neural stem cells have the potential of multidifferentiation and can differentiate into a variety of nerve terminal cells, such as neurons.
The decrease of expression of Notch 1, Hes 1 and the decrease of NICD production indicate that ATRA may directly or indirectly inhibit the differentiation of neural stem cells by inhibiting the expression of Notch 1, the increased expression of Musashi 1 and the corresponding decrease in the expression of Numb in the later period of Numb may be related to the mechanism of feedback regulation, Pr, to produce a feedback regulation mechanism, Pr. The increased expression of esenilin 1 and the increase of Rbpj expression at the later stage may also belong to the feedback regulation mechanism of.Sox 1, which may not be caused by the effect of ATRA, but because of the decrease in the expression of.Mash 1 and Neurogenin 2 expressed by the Hes 1 expression. The reduction has been reduced.
The second chapter is the role of gamma secretase in the differentiation of neural stem cells.
objective
A method based on Gal4-VP16/UAS system and double luciferase reporter gene system was established to detect the activity of gamma secretase during the differentiation of neural stem cells. The activity of gamma secretase in the differentiation process of neural stem cells, the enzyme hydrolysate NICD and the active center - Presenilin 1 (Presenilin 1, PS1) were detected for the later study of neural stem cells. The regulation mechanism of the chemical process provides an important experimental basis to detect the effect of ATRA on the activity of gamma secretase during the differentiation of neural stem cells.
Method
The Notch1 transmembrane and plasmid Notch1 Delta E-GVP of the mice were encoded with the transcription activator Gal4-VP16, encoding the luciferase luciferase gene and the plasmid MH100 of the upstream activation sequence (UAS), and the luciferase plasmid pRL-CMV of the sea kidney, and transfected into the differentiated neural stem cells by liposome transfection, and using Gal4-VP16/UAS. The system and the double luciferase reporter gene system were used to determine the activity of gamma secretase during the differentiation of neural stem cells; the production of NICD was detected by Western blot technique and the expression of PS1 in the activity center of gamma secretase was measured by real-time fluorescence quantitative PCR.
Result
Under the action of gamma secretase inhibitor DAPT, the activity of gamma secretase decreased in a dose-dependent manner and the production of NICD decreased synchronously, while the expression of PS1 in the catalytic central component increased synchronously. After the use of ATRA in the differentiated NSCs, the activity of gamma secretase increased.
conclusion
Using the Gal4-VP16/UAS system and the double luciferase reporter gene system to detect the activity of gamma secretase during the differentiation of neural stem cells, it provides an important experimental method for the study of the regulation of neural stem cells differentiation process. In the process of neural stem cell differentiation, from this regulation of the Notch pathway, ATRA The inhibitory effect of Notch pathway is on the inhibition of Notch 1 expression, not on the direct inhibition of the activity of gamma secretase and the expression of the catalytic component of the precursor protein 1.
【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

【共引文献】