【作者】 刘建军；

【导师】 蔡文琴；

【作者基本信息】 第三军医大学 ， 神经生物学， 2007， 博士

【摘要】 中枢神经系统的发育涉及包括神经诱导、细胞周期的调控、神经元特异基因的表达和神经前体细胞的分化等多个过程,而这些过程又涉及了居多信号通路以及相关基因表达产物的相互作用。总的趋势是:先由少数几个基因产物发挥作用,使部分信号通路活化,活化的信号通路又导致更多的基因转录,活化更多的信号通路;逐渐使不同的细胞出现基因表达差异,进而出现结构和功能的差异,最后细胞建立相对稳定的、可传递的、与结构和功能相适应的基因表达模式。为了解脑发育和脑损失修复过程中相关基因的表达变化,我室利用基因芯片技术对小鼠脑发育过程中基因进行筛选,获得了1037条脑发育相关基因,Dbn1是在此研究中获得的与轴突生长相关的7个基因之一。Dbn1是新发现的小鼠drebrin基因成员,Shrao T研究证实Dbn1基因位于13#染色体中间位置,由660个氨基酸组成,翻译后为120Kb大小蛋白,特异性表达在神经系统,初步研究表明其可能参与调节树突棘的形成以及神经元的迁移。Drebrin是一种与脑发育密切相关的蛋白,其特异性表达在神经系统,后来的研究显示在非神经元内也存在有drebrin的亚类表达。目前在哺乳动物发现drebrin有四种亚类,他们都是单一基因通过不同的mRNA剪接方式形成。生物信息学分析表明,drebrin有且只有一个特异的区域与actin进行结合,这个区域位于drebrin的中心,去除这个结合位点,drebrin就不能与F-actin结合。drebrin通过与actin结合调节actin细胞骨架在细胞内的聚合和解聚过程,进而引起细胞形态改变,对细胞分化迁移以及神经元突起的形成起重要的调节作用。树突形成被认为是成熟神经元突触结构可塑性最显著的特性,也是NSCs分化和迁移的重要特征和结构基础。NSCs在分化和迁移过程中,一个最主要的形态学特征改变便是突起的形成和延长,分化成熟的神经细胞在到达迁移终点后,与其周边的细胞通过树突棘建立突触(synapse)连接。树突棘是神经元树突的微小棘状突起,在脑内也是多数突触的突触后成分,其最基本的细胞骨架结构是F-actin。在树突棘内,多种actin的连接蛋白和调节蛋白伴随着轴突的生长和树突的形成而呈现不同的表达模式,他们在调节轴突和树突的形态学改变中发挥了重要的作用。因此,对细胞骨架蛋白以及相关调节蛋白的深入研究可能为解释NSCs分化、迁移等机制问题提供新的理论思路。基于Dbn1对神经元发育调节和突触可塑性的调节作用,我们推测Dbn1影响NSCs分化和迁移,对NSCs分化后突触连接的建立也具有促进作用,因此,本课题在了解Dbn1在小鼠脑发育和神经干细胞分化过程中表达模式和定位的基础上,进一步探索Dbn1对小鼠NSC体外分化的可能影响,以及分化迁移后NSC在形成突触连接过程中的作用和可能的机制。研究结果简要如下:(1)以western blot和免疫组化等方法,了解Dbn1在小鼠脑发育过程中的表达模式和定位,结果显示,在小鼠脑发育的不同阶段,均有Dbn1蛋白的表达,其中在胚胎后期,Dbn1的表达达到高峰,随后下降,出生以后表达再次上调,在出生7d时最高,随后又下降,成年以后Dbn1呈低水平表达表达;在脑内Dbn1主要表达在海马,室管膜,皮层等神经元和神经干细胞分布密集的区域;而Dbn1在细胞内的定位也有一定的时空模式,表现在胚胎时,Dbn1弥散分布在细胞的周边,而出生以后Dbn1主要分布在细胞的突起。这些结果提示Dbn1不但在脑发育发育过程中调节神经元的发生以及突触的形成,而且可能对神经干细胞的分化和迁移也具有一定影响。(2)以western blot,real-time PCR等方法,了解在NSCs分化过程中,Dbn1的表达变化模式,结果显示,在NSCs分化过程中,Dbn1有不同程度的表达,其mRNA和蛋白表达的高峰在细胞分化的第3d时,在细胞内,Dbn1主要分布在细胞的突起;免疫荧光双标显示,Dbn1与细胞内的actin蛋白有明显在共聚现象。这些结果提示:Dbn1可能调节NSCs的分化,对神经干细胞突起的形成具有一定的促进作用。(3)从出生1d的小鼠脑内扩增出Dbn1的全长基因,进而构建了EGFP-Dbn1真核表达载体,以电转法将构建的载体成功导入体外培养的神经干细胞,在无其他因子作用下,过表达Dbn1的神经干细胞能正常分化,通过免疫荧光染色,了解过表达Dbn1后,神经细胞内actin、突触后致密蛋白PSD-95和突触蛋白Synapsin1的表达变化,结果发现,在转染Dbn1以后,actin和synapsin1的表达无明显影响,而PDS-95表达水平增加,但PSD-95与Dbn1表达并不完全的共聚。结果提示在NSCs分化过程中,Dbn1与其结合的细胞骨架蛋白actin之间并无表达依赖关系,但能够调节突触相关蛋白在细胞内的表达水平,因此我们推测,Dbn1可能通过影响actin在细胞内的结构形式,进而影响突触相关蛋白在树突的表达水平。(4)根据Dbn1的基因结构特点,我们设计和构建了Dbn1 mRNA干扰载体,以病毒包装的方式干扰体外培养的神经干细胞。观察了在沉默细胞内源性Dbn1以后,细胞的分化状态和相关蛋白的表达变化,结果发现,干扰Dbn1 mRNA以后,神经干细胞能够正常分化,actin、synapsin1的表达在细胞内的表达水平也无明显改变,但PSD-95明显下降。这些结果与NSCs过表达Dbn1时,细胞状态和蛋白表达水平相适应,为我们前面的推测提供了佐证,但对于Dbn1影响神经干细胞分化过程中突触相关蛋白表达水平的确切机制还需要进一步的实验研究。更多还原

【Abstract】 The development of central nervous system (CNS) involved in neural induction, regulation of generation cycle, expression of special-gene in neurons and differentiation of precursors and so on. All these processes is associated with much of signal pathways and the reaction of many related gene products. So, the trend is as follwing: first some gene products actived some signal pathways, then the actived pathways promoted more gene transcription and actived more signal pathways; All this leads the expression difference of different genes in different cells; Finally, the gene expession pattern fitting the function of cells were established. In order to investigation the genes expression in the development of CNS , related genes were screened in our lab by High Dendity cDNA Microarrys recently. 1033 genes were hierarchical clustered and 7 genes faxilitating to axon were deteched in developmental period including Dbn1.Devolmental regulate brain proteins(drebrins) are majior actin-binding proteins in brain, and loclized at spines in adult brains. Drebrin inhibits the actin-binding activity of tropomysin and actinin, and also suppresses actomyosin interactions. There are two majior drebrin isforms:an embryonic-type isform(drebrinE) and an adult-type isform (drebrin A) in mamalian brain. A considerable data shown drebrin are dominantly expressed during embryogenesis and accumulated in neurite processes of postmigratory neurons, but the different classifications of drebrins have different temporal-spatial expressional pattern. For example, drebrinE1 and E2 mainly express in embryonic brain, but drebrin A is expressed in postnatal or adult brain. These data sugested drebrin might regulate membrane actin cytoskeletion underlies many diverse cellular events including cell motility and migration,intercellular adhesion, cell morphogenisis, and signal transduction. Dbn1 is novel nouse drebrin gene as a freshman in drebrin gene family, which mapped to the central portion of chromosome 13. expressed specificitly in nervous system and might involve in regulating formation of dendritic spine and migration of neurons.Based on the role of drebrin in regulating to neuronal developmental and synaptic plasticity, we hypothesis Dbn1 contribute to differentiation and migration, and involve in regulating to establishment of synaptic linkage after neural stem cells (NSCs) diffrentiated. In this study, we first investgated the expression of Dbn1 in developmental mouse brain and during differentiation of NSCs, furthermore, researched for the effect of Dbn1 to differentiation of NSCs by overexpression and Dbn1 siRNA. The main results were summed up as follows:(1) Develomentally, we found Dbn1 protein expressed in different develpmental stages in mouse brain, the peak of expression occurred later period of embryon(E14-E18) and down-regulating at postnatal, but the experssion of Dbn1 was increased at postnatal 7 days (P7). A lower level expression of Dbn1 was shown in adult mouse brain. In mouse brain, Dbn1 expressed majorly in hippocampus, ependymal layer and cortex,where neurons and NSCs are localized. On the other hand, location of Dbn1 in cells shown a special spatial pattern. These data suggested that Dbn1 regulat the neuron morphogenisis and formation of synapse during developing at brain, well as regulate the differentiation and migration of NSCs maybe.(2) Expression of Dbn1 mRNA and protein were investigated by real-time PCR and western blot methods. We found the expression Dbn1 mRNA and protein was highest at diffrentiated 3th days, and the most of Dbn1 protein was localized cellular membrane and prcesses. In addition, Double immunofluorescence shown Dbn1 was co-localized in the differentiated cells.these data suggested that Dbn1 may regulated the differentiation of NSCs, and promote the formation of NSCs durining their differentiation.(3) Dbn1 full gene was amplificated from P1 mouse brain. We constructed the EGFP-Dbn1 eukaryotic expression vector, and transfected the NSCs in vitro. We found the transfected NSCs differentiated normally without other factors. The expressions of actin, PSD-95 and synapsin1 were detected by immunofluorsence after EGFP-Dbn1 transfection. We didn’t find the expression of actin and synapsin1differ from control, but the expression of PDS-95 was higher than control. These results suggested there are not symbiosis between experess of Dbn1 and actin, but overexpresion Dbn1 can up-regulate expression of the synapse-associated proteins. From these,we supposeed Dbn1 regulate the expression of PSD-95 through binding actin and affected to the configuration of actin in cells.(4) According the genetic structure of Dbn1, we disigned and constructed the Dbn1 siRNA vector, and inhitited the endogenous Dbn1 of NSCs. We found the NSCs can differentiate after Dbn1 mRNA silecnce, and the experssion of actin and synapsin1 didn’t decrease compare with the control, but the expression of PSD-95 decreased obviously. These results show Dbn1 can regulate the expression of PSD-95 but not actin and synapsin1 and provide more evidence for our preceding presume during the differentiation of NSCs. Howerver, studying to the molecular mechanism about Dbn1 regulating the differentiation of NSCs and expression of synapse-associated prontein requests further empirical experiments.更多还原