The Human NR2B Gene Brian-specific Temporarily Expression In Rat Model

The N-methyl-D-aspartate (NMDA) receptor is a heteromeric ligand-gated ion channel concentrated at postsynaptic sites and involved in fast excitatory synaptic transmission and long-lasting changes in synaptic strength.NR2B is the main subunit of NMDA receptor .The Asn loop in M2 is the central of NMDA receptor which determines the pharmacological and functional properties of receptor.Blood–brain barrier (BBB) is composed of tightly connection of glial cells and capillary endothelium cells, which forms the BBB in vivo. Large-molecule drugs do not easily cross the BBB.Because of high expression of transferrin receptor in BBB,so it is a better strategy to use immunoliposome conjugated with the mouse monoclonal antibodies against rat transferring receptor(OX26), which enables targeting of the plasmid DNA or drug to the rat brain via the endogenous BBB transferrin receptor.RT-PCR was used to amplify human NR2B gene inserted into eukaryotic vector pcDNA3.1. The recombinant plasmid was transfected into the CHO, undifferentiated PC12 cells .The expression of the target molecule was confirmed by RT-PCR, Western blotting , indirect immunofluorescent staining(IFA)and the partly apoptosis was detected by flow cytometry in transfected undifferentiated PC12 when induced by NMDA receptor excitomotory, but not in transfected CHO cells.PCR was used to amplify EGFP gene and formed the recombinant plasmid pcDNA3.1-EGFP (PE). PCR was also performed based on human SynapsinI gene promoter. PCR product was inserted into PE vector excluding CMV promoter obtaining recombinant plasmid SynI-pcDNA3.1 ( -CMV ) -EGFP(SPE). Cell line CHO,COS,PC12 was transfected with the plasmids containing SPE or PE. SynI promoter can hardly drive the EGFP expression in CHO and COS,but having higher promotive effienceny in PC12 observed under fluorescent microscope and flow cytometry .We also inserted NR2B gene into SynI-pcDNA3.1(-CMV)vector obtaining recombinant plasmid SynI-pcDNA3.1(-CMV)-NR2B(SPN).Liposomes were used for encapsulating NR2B recombinant plasmid by reverse evaporation technique.They were conjugated with thiolated OX26 forming the immunoliposome. the morphology of the pegylated liposome was nearly globular, the encapsulating efficiency was about 30.4%, average diameter less than 100nm conjugated antibodies distributed uniformly around the immunoliposome. The immunoliposome could realease plasmid DNA by SDS treatment and the direct ELISA results are positive, further confirming it was immunoliposome.We divided the rats into two groups. One group is normal control,the second, third were respectively injected the immunoliposome encapsulating PN,SPN plasmid by vena caudalis. The brain including cortex and hippocampus ,Liver,Spleen were taken after 48h postinjection. the expression of exogenous NR2B gene in brain , liver, spleen were detected by RT-PCR,quantitative PCR ,western blotting, immunohischemistry. The results similarly indicated that the exogenous NR2B is expressed in both brain and Liver,Spleen when the CMV promoter is used, but only confined to the brain when the SynI promoter is used. The NR2B expression level under CMV promoter was obviously higher than SynI promoter,but the latter could specificly express in brain ,the former was extensively expressed.Tissue-specific expression in brain, silenced in other tissues, is possible after the i.v. administration of a nonviral vector with the combined use of immunoliposome and tissue-specific promoter.The NR2B gene brian-specific temporarily expressionin rat model could search for effective method for gene therapy of the NR2B such as learning, memory processing, and nerves disorders; also for effective DNA delivery system.