| Background Ischemic cerebrovascular disease is a kind of common and frequent disease that severely imperils patients' lives. Many studies have been done for the disease both at home and abroad, which gradually deepens the comprehension of the ischemic injury and improves the prognosis. However, an ideal curative effect has not been achieved as yet. So it is necessary to search for a new and a stonger pointed therapy strategy. Brain ischemia/reperfusion has relationship with many factors, such as free radicals, the excitotoxicity, calcium over-loading, the dysfunction of mitochondria, inflammation and apoptosis. A good body of studies suggest the inflammatory response during reperfusion speeds the secondary brain damages and play a key role in I/R. While cerebral microvascular endothelial cells' injury and dysfunction is a key event early in the pathogenesis of cerebrovascular disease, so it is important to protect the function and stucture of endothelial cells from injury. The activation of endothelial cells is a critical step on the onset of inflammatory responses and is associated with the up-regulation of a variety of adhesion molecules and cytokines under the regulation of NF-κB. The interactions of leukocyte-activated endothelial cells make tissue injury. Many scholars hold that the NF-κB/ IκB system may play a key role in inflammatory responses. NF-κB decoy strategy has been a powerful tool for gene therapy and gene regulation in the study of transcriptional regulation. NF-κB decoy ODNs in dumbbell shape with the nuclear sequence which was similar to NF-κB cis-elements were synthetized in our study. Oligodeoxynucleotides with high affinity for transcription factors may be used to bind the transcription factors and block the activation of target gene. However, problems such as the short circulation time, the poor stability of these oligodeoxynucleotides versus nuclease activity and the low intracellular penetration were discovered. The aftereffect was high cost and high cytotoxicity. And it's known that blood-brain barrier limited many drugs and oligodeoxynucleotides to enter the brain. So it's urgent to develop a new carrier that can delivery drugs across BBB and then exert some therapeutic effects. Nanoparticles, as a kind of targeting carrier, have attracted many attentions. When NP were modified, they could improve the stability against nuclease and enhance decoy ODNs intracellular penetration. Therefore NP have the potential to carry genes acroos BBB, target the brain and then exert curative effects for CNS diseases. Objective (1) To isolate and to observe the morphology of brain microvascular endothelial cells (BMEC) from Wistar rat. (2) To prepare cationic polybutylcyanoacrylate nanoparticles (PBCA NP) with uniform size, high gene loading capacity and high transfection efficiency. (3) To establish a simple, stable and reliable model of oxygen glucose deprivation/reoxygenation (OGD/R) of vascular endothelial cells in vitro. (4) To investigate the protein expression and the activation of NF-κBp65 following OGD/R of BMEC in vitro. (5) To investigate whether NF-κB decoy oligonucleotides(ODNs) -PBCA NP could affect the protein expression of intercellular adhesion molecule-1 (ICAM-1) and inducible nitric oxide synthase (iNOS) following OGD/R in BMEC. Methods BMEC of Wistar weanling rats were prepared by filtering through a mesh, centrifugation and collagenase digestion. The BMEC were affirmed by immunocytochemical method, morphologies under Light Microscope. We observed the growth curve of BMEC by MTT assay. We use transmission Electron Microscope(EM), Zetasizer4700, laser diversity particle sizer, flow cytometry, fluorescent microscope to characterize the PBCA NP's properties, the mean diameter, the genes loading capacity and its ability of brain targeting. Cells injury in the model of OGD was evaluate by the trypan blue test, MTT assay, LDH release. Morphologic changes were studied under Light Microscope(LM) and EM. We studied the activation, the nuclear translocation of...
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