The Correlational Research Of Pre-B-cell Colony Enhancing Factor(PBEF) Increasing Endothelial Permeability In Hypoxia/Reoxygenation

Objective: To establish hypoxia/reoxygenation model, and detect the proteinexpress of pre-B-cell colony-enhancing factor in endothelial cells after hypoxia/reoxygenation.Through RNA interference silencing PBEF gene so as to explore therelationship between PBEF and VEGF and p-MLC, we can find the mechanism ofPBEF increasing permeability of endothelial cells in hypoxia/reoxygenation.Metholds:1、Based on the literature to synthesis PBEFsiRNA. According to thedifferent treatment for HUVEC, it can be divided into empty liposome group,negative control group and PBEFsiRNA group. So we can analyze the gene level andprotein level of PBEF though RT-PCR and Western blot.2、To establish endothelial cells hypoxia/reoxygenation model, according to thedifferent HUVEC treatment, it can be divided into normal control group, hypoxia (20hours) and reoxygenation (3h) group, hypoxia (20h) and reoxygenation (6h) group,hypoxia (20h) and reoxygenation (9hours) group, hypoxia(20h)/reoxygenation (12h); The expressions of PBEF ware tested by Western blot.3、According to the different treatment，HUVEC were randomly assignedequally to: group Ⅰ (hypoxia/reoxygenation+empty liposomes group), group Ⅱ(hypoxia/reoxygenation+negative control group), group Ⅲ (hypoxia/reoxygenation+PBEFsiRNA), group Ⅳ (empty liposomes control group).Theexpressions of VEGF and p-MLC were determined by Western blot.Results:1、The mRNA and protein expression of PBEF in PBEFsiRNA groupwere significantly lower compared to liposome group and the negative controls (P <0.05), and the difference between the negative control group and liposome group hadno statistical significance (P>0.05).2、The expression of PBEF protein in hypoxia/reoxygenation group wassignificantly highter than the normal control group.It increased in the3h ofreoxygenation group, peaked at9h, until12h started to decline.(P<0.05)3、After treating HUVEC with hypoxia/reoxygenation and RNA interference,the VEGF, p-MLC expression of group I and group II was significantly highter than group Ⅳ(P <0.05), and group III was significantly highter than group I and groupII(P <0.05); The VEGF, p-MLC expression between group Ⅰ and Ⅱ group werenot significantly different (P>0.05).Conclusions:1、The expression of PBEF, VEGF and p-MLC were significantlyhigher in endothelial cell after Hypoxia/reoxygenation.2、When the PBEF gene was knockdown, the expression of VEGF in hypoxiaand reoxygenation is significantly lower, indicating that PBEF may change thepermeability of endothelial cells by regulating the expression of VEGF.3、When the PBEF gene was knockdown, the expression of p-MLC in hypoxiaand reoxygenation is significantly lower，indicating that PBEF may plays a importantrole in cytoskeletal changes by regulating the phosphorylation of MLC.