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The Mechanism Study Of NF-κB Decoy Strategy Resisting The Inflammatory Injury Following Oxygen Glucose Deprivation/Reoxygenation In Vascular Edothelialcells

Posted on:2005-07-21Degree:MasterType:Thesis
Country:ChinaCandidate:B ChenFull Text:PDF
GTID:2144360122490053Subject:Neurology
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Background A growing body of evidence suggests that the NF-KB/ IkB system may play a key role in inflammatory responses which involved in infection,ischemia/reperfusion and arteriosclerosis. The activation of endothelial cells is a critical step in 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-KB. The interactions of leukocyte-activated endothelial cells make tissue injury.NF-KB decoy strategy has been a powerful tool for gene therapy and gene regulation in the study of transcriptional regulation. It may be more effective than other strategy. Oligodeoxynucleotides with high affinity for transcription factors may be introduced in as a vitro decoy cis element to bind the transcription factors and block the activation of targetgene.Objective (1) To establish a simple, stable and reliable modle of oxygen glucose deprivation/reoxygenation (OGD/R)of vascular endothelial cells in vitro. (2) To investigate the protein expression and the activation of NF-KB p65 following OGD/R of ECV304 cell strain in vitro. (3) To investigate whether NF-KB circular dumbbell DNA oligonucleotides (CDODN-KB) could affect the protein expression of intercellular adhesion molecule-1 (ICAM-1) and inducible nitric oxide synthase (iNOS) following OGD/R in ECV304 cells.Methods 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 transmission Electron Microscope(EM). We studied the activation, the nuclear translocation of NF-KB p65 and the protein expression of ICAM-1 and iNOS in each group ECV304 cells by immunocytochemical method. We used EMS A to detect the NF-KB binding activity of CDODN-kB.Results The levels of oxygen concentration,oxygen saturation, oxygen parial pressure were very low during the OGD. There were significant changes in morphology and ultrastructure of ECV304 cells before and after OGD 4h under LM, EM. The pattern of ECV304 cells injury indicated time-dependent through trypan blue test, MTT assay, LDHrelease. Treated groups had significant differences compared with normal control group(P<0.05), The protein expression of NF-kB p65 increased during the OGD/R. It reached its peak after treated 4h. The number of positive cells with NF-KB nuclear translocation increased after OGD4h and reached its peak at reoxygen 4h group, which had significant differences compared with each group(P<0.05). The FAM-labeled CDODN-KB was observed after 6h transfected by cationic liposome into ECV304 cells under FM.EMSA showed CDODN-KB but not CDODN-kB-scr had high affinity to bind NF-KB. The protein expression of ICAM-1 and iNOS in CDODN-KB group ECV304 cells treated by OGD4h/R6h decreased, but CDODN-KB-scr group did not through immunocytochemical method. The HSCORE of protein expression of ICAM-1 and iNOS of CDODN-KB group has significant difference compared with the control group at OGD4h/R6h (P<0.05).Conclusions We have established a simple, stable and reliable modle of OGD/R of vascular endothelial cells in vitro successfully. NF-KB of ECV304 cells was actived quickly when treaed by OGD. The protein expression of NF-KB reached its peak from OGD 4h to OGD4h/R4h. EMSA showed CDODN-KB has high affinity to bind NF-kB. The FAM-labeled CDODN-KB was transfected by cationic liposome intoECV304 cells nuclei successfully. The NF-kB decoy CDODN-kB blocked OGD4H/R6 induced ICAM-1 and iNOS upregulation in ECV304 cells. It demonstrate that the NF-KB decoy could modulate inflammatory mediator in vascular endothelial cells after OGD/R. The NF-KB decoy may be of potential pharmacological use for cerebral ischemia/erperfusion injury.
Keywords/Search Tags:NF-κB, oxygen glucose deprivation/reoxygenation, vascular endothelial cells, decoy strategy, circular dumbbell DNA oligonucleotides, ICAM-1, iNOS
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