The Study Of Relationship And Effect Between Homer1a And Notch Signal Pathway After Mechnical Neuronal Injury In Vitro

Notch genes was first found in Drosophila in1917,and it got this namejust because Partially missing of its functions can cause Notch of fly wingedge. Notch signaling pathway is an conservative intercellular signaltransduction pathway, which plays a very important role in many Central andperipheral tissues and organs during early development,and the preciseregulation of this signaling pathways is essential for the normal developmentof most organizations. Past literature shows a transient upregulation ofexpression of Notch signaling pathway after freezing damage in brain tissue.Scholars speculate that the Notch Signaling Pathway may play an importantrole in brain injury. Homer proteins family were discovered for the first timein1997. Homer proteins,which is the key point of cell signal transductionsystem, involved in the transport of mGluR1a from intracellular endoplasmicreticulum to the postsynaptic membrane,and it also affects the functions ofCa2+channel, Shank,and cytoskeletal proteins. Past literature shows thatHomer protein plays an important role in various signaling pathway such ascellular swelling, necrosis, apoptosis and regeneration. This experiment study on interaction rules between Notch signaling pathway and Homer-1a proteinin cultured neuronal cells after mechanical injury by usingImmunohistochemistry and Western blot methods, and preliminary discussionon its meaning, which may lay a foundation for the next exploration onmolecular mechanism of traumatic brain injury.Part Ⅰ mechanically injured model of cultured cortical neuronsAbstract Objective Establish and validate mechanical injury model inprimary cultured cortical neurons. Methods Using fetal mice to primaryculturing cerebral cortex neurons and observating their growth by microscope.Scratching neurons with shifting liquid gun head after7days to matureneurons and lead to mechanical damage. Observating cell morphologychanges and detecting lactate dehydrogenase activity(LDH) to evaluateneuronal damage and verify the feasibility of the model. Results The neuronalnucleus was observed with round outline, and it was large and clear.With theuseage of microscope, the membrane of nucleus and the nucleolus couldeasily found.Many big-figure neurons was observed, with bright cytoplasmand many dendrites and axons connected to each other,which form acomplicated and special reticular pattern. After process of mechanical injury,scratch area neuron structure completely disappeared. More visible cell debrisdamaged cell processes around the damaged area. Lactate dehydrogenaseactivity in culture medium is elevated. Conclusion Scratch model is asimple and efficient mechanical injury model of neurons cultured in vitro.Part Ⅱ Distribution and expression trends of NICD and Homer-1a afterthe mechanical injuryAbstract Objective Observating the distribution and expression of trends ofNICD and Homer-1a in the normal and Scratch model in vitro. Methods Using Immunohistochemistry method to testing distribution of NICD andHomer-1a on mature neurons. Using Western Blot method to detectingexpression of Homer-1a and NICD after mechanical injury. Results NICDand Homer-1a are coexpress on the bodies and bumps of Mature neurons,andthey show a certain expression trend respectively at different points in time(1h,3h,6h,12h,24h) after mechanical injury. Conclusions NICD andHomer-1a are coexpressed in mature neurons, and they possibly play a role inmechanical injury in neurons.Part Ⅲ Effects of change of Notch Signaling on neuronal mechanicalinjury and Homer-1aAbstract Objective Observating the effects of change of Notch Signaling onneuronal mechanical injury and Homer-1a our study. Methods Using DAPTand VPA pretreat neurons24h respectively, after mechanical injury ofneurons, then Western Blot detection was used to anlysising the expression ofCaspase-3and Homer-1a. Results Compared with the injury group aftermechanical injury, DAPT group show a significantly lower expression ofCaspase-3(P<0.05) and Homer-1a (P<0.05), while VPA group show asignificantly elevated expression of Caspase-3(P<0.05) and Homer-1a(P<0.05). Conclusions Inhibition of Notch Signaling reduces neuronalapoptosis after mechanical injury while upregulation of Notch Signaling canincrease neuronal apoptosis after mechanical injury, this effect may ralate tothe regulation of Notch Signaling on Homer-1a.Part Ⅳ Effects of Overexpression of Homer-1a on neuronal mechanicalinjury and Notch SignalingAbstract Objective Observating the effects of overexpression of Homer-1a onneuronal mechanical injury and Notch Signaling. Methods Using virus vector pretreat neurons24h to overexpressing Homer-1a. Western Blotdetection was used to anlysising the expression of Caspase-3and NICD.Results Compared with the injury group after mechanical injury,overexpression group show a significantly elevated expression ofCaspase-3(P<0.05),but has no effect on NICD (P>0.05). ConclusionsOverexpression of Homer-1a increased the neuronal apoptosis aftermechanical injury. Homer-1a may be in the downstream of Notch Signaling.