The Effect Of Propofol On Oxygen Glucose Deprivation Hippocampus Neuron

ObjectiveTo explore the effect of differental concentration propofol on primary cultural hippocampus neuron undergo oxygen glucose deprivation,and to provide pilot evidence for neuronal protective effect of propol.Methods1.Treat methods:Put primary cultural hippocampus neuron into defferental trial groups randomly,C group(only OGD),D group(OGD+DMSO),P10(OGD+propofol 10μM),P50 group(OGD+propofol 50μM),P100 group (OGD+propofol 100μM).Oxygen glucose deprive them for 60min by removing the original glucose-containing medium and replacing with a glucose-free balanced salt solution,then all dishes were placed in an anaerobic chamber(95%N₂+5%CO₂).The cells are collected and extracted total RNA at 1h,6h,12h,24h,48h,72h and168h after OGD.2.Detect the level of HO-1mRNA:the first,RT-PCR;secondly,the semi-quantitative measure of mRNA expression was expressed as the ratio of integrated optical density(IOD) with HO-1/β-actin.3.Other detected methods employed in present research:To assess viability of cultured cell by Trypan blue staining,and to estimate the propotion of neurons and glias by immunocytochemical staining with neurone specific enolase.Results:1.The cultural method of hippocampus neuron and evaluation about purity of neuron:dissociate hippocampus tissue for 8 minute by 0.25%trypasin;after culture 6h with serum-supplemented media,change it with serum-free media,add 5μmol/L arabinosylcytosine to media at 48h and maintain 24h,then go on culturing;the evvaluation of neuron purity is above 90%by NSE. 2.Statistic outcome(ANOVA) show that there is significant difference among each group(P＜0.05).longer of the OGD time,lower of the cell survival rate.3.The optimized system and conditions of PCR:templetes 2μl,10×buffer(contain MgC12 1.5mmol/L) 2.5μl,dNTP(10mM) 0.5μl,Taq DNA polymerase 0.5μl,upper and lower primer 0.5μl respectively,the total volum was 25μl;After 5 min at 94℃,reactions were cycled through 30sec denaturation at 94℃,30sec annealing at 52℃,and 40sec extension at 72℃for 26 repeats,followed 72℃for 5 minutes.4.Outcomes based on two-way ANOVA show that:a.There is time trends about HO-1 mRNA of hippocampus neurons that were OGD at different time point(P＜0.05);b.There isn't significant difference on HO-1 mRNA of hippocampus neurons after OGD with or without propofol and from 10μM to 100μM(P＞0.05).Conclusions:1.Propofol can't change HO-1 mRNA level of hippocampus neuron which subjected oxygen-glucose depriviation.2.It is necessary that resaerch the change of HO-1 protein to elucidate the effection about propofol to expression of HO-1 in hippocampus neuron.