A Study Of The Protection And The Mechine Of Brain Derived Neurotrophic Factor To Cortical Neurons

PART 1 OBSERVATION OF CORTICAL NEURON IN VITRO USING MICROSCOPEObjective:To explore the methods of culture of cortical neuron from neonatal rat,and observe ultrastructure of the cortical neuron.Methods:To isolate the cerebral cortex from Sprague Dawley rats of 0-12 hours old using mechanical seperation,scissors inro pieces and digest using trypsin,culture cortical neurons after centrifuging using 10% fetal calf serum.change into Dulbecco's modified eagle medium/F12 containing 2%B27 in 12 hours,change cell culture medium every three days,obeserve the ultrastructure of cortical neurons with microscope when it was culture 7 days.Results:Cortical neurons take on globalar when just be inoculated, the cell begin to stretch out short axon in 4 hours,cell body become bigger,and have halation,solid.axon become more thickening than before,and axons erect initial connection after 12 hours,when Cell grow up to the 7 days,Cell body become more and more big,neuron axons transform into thickening than before,the connection between axons become to more tighter,and already eatablish compact pykno-lattice.Part 2 Brain derived neurotrophic factor can protect cortical neurons from L-glutamic acidObjective:To explore brain derived neurotrophic factor can protect cortical neurons from L-glutamic acid.Methods:Set up control group,BDNF group,glutamic acid control group is cortical neurons cultured as usual,BDNF group is adding 50 ng/ml BDNF into cell medium of the fifth day's cortical neurons for 24 hours,after 24 hours ,transform the cell medium into DMEM/F12 containing 50μmol/L glutamate for 30 minutes,then culture the cell using the virgin cell medium,and the glutamic acid control group is only add 50μmol/L glutamate for 30 minutes on the sixth day.Results:The control group:cell nucleus present green fluorescence,and morphous integrity,axon clear.the BDNF group:some cell nucleus membrane fracture,we can see red fluorescence in the cells,some axons shrinkage.the Glutamic acid group:the cell with red fluorescence increase more. The mortality of neurons in BDNF group(1.14±0.06)is lower than in the control group(0.72±0.10) (P<0.01)Conclusion:BDNF can protect cortical neurons.Part3 The mechanism of BDNF protect cortical neuron aginst glutemateObjective:The mechanism of BDNF protect cortical neuron aginst glutemateMethods:Primary cultured cortical neurons cell from Sprague Dawlay rats of newborn were cultured for 8d.The cortical neurons were divided randomly into three groups:control group,Glu group and BDNF group.detect the quantity of the protein by western-blot. pression of p75NTR,JNK,ERK were obeserved using western blot analysis.Results:At the 8 day,the primary cortical neurons grew well,BDNF can protect cortical neurals cell from glutamate.the expression of p75NTR is higher in control group(0.90±0.18)and BDNF group (0.78±0.09)than in nomal group(0.15±0.12)(P﹤0.05);the expression of JNK increase in the control group(0.95±0.06)to the BDNF group(0.56±0.16)(P﹤0.05);at the same time,the expression of ERK decrease in the control group(0.57±0.08)to the BDNF group(0.83±0.16)(P﹤0.05).The difference between the control group and the BDNFgroup have statistical significance.Conclusion:BDNF can adjust the proportion between the p75NTR and the TrkB to protect the cortical neurons aginst neurotoxicity induced by glutamate.In addition,enhance the survival and suppress the apoptosis by increasing the expression of the ERK downstream signal and decreasing the expression of the JNK downstream signal.