Effect Of Oecs Over-expressed EsRAGE On AGE-induced Inflammation Reaction In RMECs

Objective:Endogenous secretory receptor for advanced glycation end products (esRAGE)is a kind of secretory receptor for advanced glycation end products(sRAGE), couldcompete bind with the advanced glycation end products (AGEs), block AGE-RAGEpathway, reversing the development of inflammation.OECs belonging to lateendothelial cells, also called mature endothelial cells, have potential repair functionininflammatory retinal vascular,and also as a good gene carrier.With this study, OECsbe used as a carrier of esRAGE gene, while increasing the esRAGE gene expressionof OECs, repair the demaged blood vessels and block the AGE-induced inflammatoryresponse.Methods:Construct overexpression adenoviral vectors of esRAGE gene. Collect fresh SDrat peripheral blood mononuclear cells(MNCs) and MNCs were obtained by densitygradient fractionation, OECs have been identified by phenotype. Put theover-expression esRAGE gene adenovirus vector transfer to OECs, identified thegene expression in cells. Then,with AGE-BSA induced rat retinal microvascularendothelial cells inflammatory changes, put the OECs that have been transfectedesRAGE co-cultured with inflammatory RMECs, observe retinal cells expression ofinflammatory cytokines.TUNEL assay to identify AGE-induced RMECs cellstransfected group and the Ad-esRAGE-OECs with AGE-induced apoptosis ofcultured cells,Clear the virus and OECs influence to RMECs apoptosis that inducedby AGE.Result:1. Builds successfully over-expression esRAGE adenovirus vector.2.Endothelial cell surface markers of CD34, CD133and VEGFR-2streamingantibodies to identify OECs cells and streaming results were5.41%,3.45%and91.50%respectively.Prompt successful isolation and culture OECs cell. 3.RTQ-PCR results showed that transfection esRAGE adenovirus group almost13times higher than that of control group;Western-blot results showed that theprotein expression of esRAGE in experimental group,and the control group did notsee esRAGE protein stripe;ELISA results showed that the experimental groupexpression quantity almost6times higher than the control group.Ad-esRAGEsuccessed into OECs and high expression esRAGE in cells and esRAGE withextracellular secretion.4.RMECs cell immunofluorescence results showed, DAPI stain nuclei into blue,VIII-FITC performance for the green, and the cell membrane and cytoplasm appeargreen fluorescent, showed that factor VIII antigen main expression in the cellmembrane and cytoplasm.Prompt successful isolation and culture RMECs cell.5.AGE-BSA concentration to100ug/ml of24h, the NF-κB expression increasedsignificantly (P <0.01), RGAE and inflammatory cytokines VEGF, TGF-β, MCP-1expression increased of different degrees, and show the dose-and time-dependent.The AGE-BSA concentration to100ug/ml of12h, compared with control group, thedifference was statistically significant (P <0.05).6.Confocal results showed green fluorescence labeled RMECs and redfluorescence labeled OECs can be merge and integrated to form a tubular structure.7. RTQ-PCR and Western-blot results showed that transfected with Ad-esRAGE-OECs cells role in AGE-induced RMECs cells, RAGE of RMECs cell andinflammatory factor NF-κB,VEGF, TGF-β, MCP-1mRNA and protein expressiondecreased obviously, the difference was statistically significant (P <0.05).8.The apoptosis of AGE-induced RMECs inflammatory cells group comparedwith Ad-esRAGE-OECs cell group, there was no statistically significant difference(P>0.05).Conclusions:1.OECs can be used as a carrier of esRAGE gene, OECs transfected withesRAGE gene have the effect of extracellular secretion.2.AGE can induce inflammation in RMECs, and significant increase RGAE andinflammatory cytokines VEGF, TGF-β, MCP-1expression.3.OECs that transfected gene secretion of esRAGE can significantly reduce the AGE-induced RMECs inflammation,significantly reduce RGAE and inflammatorycytokines VEGF, TGF-β, MCP-1expression.4.OECs that transfected esRAGE gene can be fused into RMECs vasculature andto play its role in the repair of blood vessels, while not increasing apoptosis ofendothelial cells.