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The Role Of DIP2A On Neurite Outgrowth In Cultured Hippocampal Neuron

Posted on:2018-09-17Degree:MasterType:Thesis
Country:ChinaCandidate:C SuFull Text:PDF
GTID:2404330515969442Subject:Cell biology
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Cerebral development is the basis for the formation and establishment of neurological function in mammals.The process includes the proliferation,differentiation,migration,neuronal axon and dendritic growth and synaptic establishment of neural precursor cells.The neuronal growthshow that neuronhas a very long axon and multiple dendrites.Neurons are the basis for the transmission of information,receiving information and processing information in the nervous system,establishing synaptic links and ensuring the function of the nervous system.Autism has a variety of symptoms,most of the patients in the first three years have brain development problem during the critical period of the formation,non-functional abnormalities can be used as a sign for clinical diagnosis,including abnormal neurite outgrowth.DIP2 A is a highly conserved protein,in the bioinformatics analysis DIP2 A wasfound to be involved in the development of the nervous system.DIP2 A is a susceptible gene for dyslexia and autism.In Drosophila neuron DIP2 can regulate the branch and orientation of axon of mushroom neuron.The homology of DIP2 gene in mice and Drosophila melanogaster is high,which provides a foundation for us to study the effect of DIP2 A on neurite outgrowth.So we want to solve the problem is whether DIP2 Aaffect the growth of the protrusions to promote the formation of autism in the development of the nervous system.In this study,we use the method of cultured hippocampal neurons in vitro as the main experimental method to study the function of DIP2 A in the neuron.Immunofluorescence staining in the hippocampal neuron cultured for 3 days in vitro showed that endogenous DIP2 A is expressed in soma,axon and dendrite in neuron,using axon marker Tau-1 and dendritic marker MAP2,respectively.We also isolated the hippocampal neurons from the P0 mice in the same litter-matched DIP2A-knockout mice and wild-type mice.When cultured for four days in vitro,axons are labeled by Tau-1.When cultured for seven days in vitro,dendriteis labeled by MAP2.It is found that axons from DIP2A-knockout mice do not show significant abnormalities,but the dendrite show the growth development defect.Then we transfect the neurons using the plasmid EGFP and DIP2A-EGFP,respectively,and labeled in the same manner,it is found that overexpression of DIP2 Ado not show obvious axon abnormalities,but the development of dendrites show obvious outgrowth.At the same time,we construct different domain to the Flag plasmid to rescue this kind of phenotype.Then we transfect different domains from DIP2 A to rescue the dendrite phenotype fromDIP2 A knockout mice.It is found that the DMAP-CaiC domain from DIP2 A play a critical role in the normal growth and abundance of dendrite.In conclusion,DIP2 A is selective for the role of neurite.DIP2 A has no effect on the growth and development of axon,but it is important for the growth and development of dendrite,suggesting that neurons may be abnormal in the process of receiving information and processing information.And DMAP-CaiC domain is important for dendritic growth,providing a reference for the pathogenesis of autism.
Keywords/Search Tags:DIP2A, neurite outgrowth, axon, dendrite, rescue
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