| 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.
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