The Expressional Changes Of AMPA Receptors In Hippocampal Neurons Transfected With Lentiviral Gene Silencing AP4M1

ObjectivePrimarily,hippocampal neurons of fetal rats were cultured,and then were transfected into lentivirus,which downregulated adaptor-related protein complex 4,mu 1 subunit(AP4M1)gene.The purposes of this study were to investigate the expressional changes of AP4M1 and AMPA receptor,in order to explore the mechanism of AP4M1 mediated AMPA receptor changes.MethodsIn this study,primarily cultured hippocampal neurons of fetal rats were selected as the experimental subjects.We identified the purity of neurons through immunofluorescence by labeling the neuronal specific enolase(NSE)on neurons.Neurons were transfected with lentivirus and then were determined the transfection efficiency by fluorescence detection.The experiment was divided into negative lentivirus transfection group and positive lentivirus transfection group(silent AP4M1 gene).The expression of mRNA and protein of AP4M1 and AMPARs were detected by Real-time PCR and Western blotting.Result1.Primary culture,identification and transfection efficiency of hippocampal neuronsPrimarily cultured hippocampal neurons of SD fatel rats were grown well,and they developed mature on the 7th day in vitro.The results of immunofluorescence staining showed that the purity of neurons was more than 95%.Lentivirus was transfected on day 2,and lentivirus was replaced after 8-12 hours,and then the above transfection method was repeated again.The transfection rate was greater than 85%.2.Variation of AP4M1,AMPARs in hippocampal neurons which were transfected with lentivirus1)Real-time PCR: The variation of AP4M1 mRNA in the positive virus group was significantly lower than that in the negative virus group.The expression of AP4M1 mRNA was lower than that of the negative virus group(F = 19.980,t =68.193,p <0.01,n = 8)).The expression of GluR1 mRNA was lower than that of the negative virus group(F = 348.400,t = 8.060,p <0.01,n = 8).2)Western blotting: The expression of AP4M1,GluR1 and GluR2 was downregulated(F = 7.566,t' = 15.966,p <0.01,n = 8)<0.01,n = 8),(F = 0.408,t '=8.690,p <0.01),(F = 5.560,t '= 13.770,p <0.01,n = 8).Conclusions:The mRNA and protein expression of AP4M1 in hippocampal neurons were significantly down-regulated after transfection with lentivirus with silent gene AP4M1,indicating that transfection was effective and the mRNA and protein expression of GluR1,2 in hippocampal neurons were also down-regulated;AP4M1may directly or indirectly regulate the transport of GluR1,2 in hippocampal neurons.