内毒素诱导血管内皮细胞nf-κb-jmjd3信号轴下游基因表达的表观遗传学调控机制-epigenetic regulation mechanism of nf - κ b - jmjd 3 signal axis downstream gene expression induced by endotoxin in vascular endothelial cells.docxVIP

Abst Abstract IV IV ABSTRACT Objective Chronic inflammation communed closely with cancer, of which vascular endothelial cells play a key role in the pathological process. The research was designed to observe the inductive effect of inflammatory mediators and adhesion molecules in LPS induced vascular endothelial cells, and to explpore the epigenetic mechanism of NF-κB- Jmjd3 signaling pathway, which will provide reliable experimental basis for effective inhibition of inflammation in vessels and prommising target therapies of related diseases. Methods The primary human umbilical vein endothelial cells were thawed and cultured routinely, then divided into differrent LPS groups randomly by processing with LPS for certain time course according to the demand of each experiment, control group was set simultanously. The concentration of IL-6, MMP -9 and ICAM -1 in supernatants were detected by ELISA kits to identify the release of inflammatory mediators and adhesion molecules induced by LPS in endothelial cells. The expression and location of NF-κB/p65 and Jmjd3 were observed by immuno-fluorescence technique, and RNA levels of Jmjd3 was determined by RT-PCR，then the expression of NF-κB/p65, IκBα, and Jmjd3 were verified by Western blotting. Activity of transcription factor NF-κB in nuclear extraction was detected by Transcription Factor Assay Kit of Millipore. Recruitment of NF-κB/p65, Jmjd3 and H3K27me3 along transcription start binding sites of target genes was analyzed by chromatin immunprecipitation technique: the targeted DNA library was build by Chromatin Immuno-precipitation kit of Millipore, following ChIP-qPCR to analyze NF-κB/p65, Jmjd3 and H3K27me3 recruitment along transcription start binding sites of target genes PAGE PAGE VI including IL-6, MMP-9 and ICAM-1. Results The release of inflammatory mediators and adhesion molecules in LPS induced HUVECs turned out to be that the relative concentration of IL-6, MMP-9 and ICAM-1 were up-regulated