The PIM Expression Of Endothelial Progenitor Cells Induced By Shear Stress And VEGF Mediated By ERK1/2 Signal Molecule

Objective:To investigate the effect of mechanical factors(fluid shear stress,FSS)and chemical factors(vascular endothelial growth factor,VEGF)on the PIM kinases family in endothelial progenitor cells(EPCs)and its possible signaling mechanism.Methods:(1)Isolation,culture and identification of human umbilical cord blood endothelial progenitor cells:density gradient centrifugation combined with differential adherence method to isolate human umbilical cord blood endothelial progenitor cells;phagocytosis of Dil-acLDL and binding to FITC-UEA-I ability to carry out cytological identification.(2)The effect of VEGF on the expression of PIM kinase family:real-time quantitative PCR was used to detect the expression level of PIM kinase family at 0.5 h,1 h,2 h,4 h and 6 h after VEGF stimulation.(3)Detection the PIM kinase family gene expression: analyze the level of PIM under different shear stress(low 5 dyne/cm2,medium 15 dyne/cm2 and high 30 dyne/cm2)and at different time points(1 h,2 h,4 h and 6 h)by real-time quantitative PCR.(4)The addition of ERK1/2 specific inhibitor PD98059 significantly inhibited the up-regulation of Pim-1 by VEGF and the up-regulation of Pim-1,Pim-2 and Pim-3 by shear stress.(5)Detection the changes related signaling molecules p-ERK1/2 under 5dyne/cm2 shear stress by Western Blot technique.Results:(1)The morphology and identification of human umbilical cord endothelial progenitor cells: endothelial progenitor cells are easy to cluster,the middle cells are round,the edges are spindle shape,and the individual cells are mostly spindle.phagocytosis of Dil-acLDL and binding to FITC-UEA-I cells showed red and green respectively in the cytoplasm.(2)The expression of Pim-1 was significantly up-regulated under VEGF,but had no effect on Pim-2 and Pim-3.(3)The expression of the Pim-1,Pim-2 and Pim-3 were all up-regulated in the PIM kinase family under shear stress intervention,but Pim-1 and Pim-2 were enhanced obviously at low shear stress,while Pim-3 was up-reg?Lated more obviously at medium shear stress.(4)The addition of ERK1/2 specific inhibitor PD98059 significantly inhibited the up-regulation of Pim-1 by VEGF,and also inhibited the up-regulation of Pim-1,Pim-2 and Pim-3 expression induced by shear stress.(5)At short-term phase of low shear stress,P38,ERK1/2,Akt and STAT3 phosphorylation were changed;in short tangential stress gradient,phosphorylation of ERK1/2 showed a tendency to increase first and then decrease gradually.Conclusions: VEGF could up-regulate the expression of Pim-1,and the shear stress could significantly up-regulate the expression of Pim-1,Pim-2 and Pim-3,and ERK1/2 signaling may be involved in this up-regulation.