Singnaling Pathway In The Differentiation Process From Hadscs Into LECs

ObjectiveTo explore the importance of VEGFR3 and Integrinα9 in the diff erentiation process from human adipose-derived stem cells(HADSCs) in to lymphatic endothelial cells(LECs) by vascular endothelial growth fact or C(VEGF-C156s) and migration of induced cells.Methods(1)HADSCs were randomly divided into 3 groups:VEGF-C ind ucing group(containing 100ng/ml VEGF-C)、VEGF-C unite VEGFR3 phosphorylase inhibitor group(containing 100ng/ml VEGF-C+5umol/L VEGFR3 phosphorylase inhibitor MAZ51)、blank control group(conve ntional DMEM/F12 medium),after 10 d inducing,the phosphorylated VE GFR3(p-VEGFR3) were tested with Western blot.(2)Another HADSCs were randomly divided into 5 groups: bl ank control group、VEGF-C inducing group(containing 100ng/ml VEG F-C)、VEGF-C unite inhibitor “a” group(containing 100ng/ml VEGFC+5umol/L VEGFR3 phosphorylase inhibitor MAZ51)、VEGF-C unite inhibitor“b”group(containing 100ng/ml VEGF-C+10ug/ml Anti-Integrinα9 function blocking antibody Y9A2)、VEGF-C unite inhibitor“a”and“b”group(containing 100ng/ml VEGF-C+the two kinds of inhibitors with the ditto concentration).After 10 d culturing,the relative expression l evel of LECs markers VEGFR3、Integrinα9、LYVE-1 were detected by immunofluorescence technique and Western blot.(3)After 10 d inducing with 100 ng/ml VEGF-C,the cells were i ncubated by 30ug/ml Anti-Integrinα9 function blocking antibody、5umol/L VEGFR3 phosphorylase inhibitor and united the two inhibitor above,set the control group,all groups, quantity of cell migtation in all grou ps were cauculated using Transwell.Results(1)5umol/L VEGFR3 phosphorylase inhibitor reduced the expres sion level of p-VEGFR3 significantly.(2)Compared with that in induction group, the expression level of LYVE-1 was modestly weakened with blocking VEGFR3 and Int egrinα9 pathways(PWestern=0.000,PIF=0.000), and there are no statis tical difference in LYVE-1 level between the two inhibitors at the co ncentration above(PWestern=0.164﹥0.05,PIF=0.927﹥0.05);Blocking o ne of the two pathways, the expression of another had no change(P WesternVEGFR3(BVSD)=O.804,PIFVEGFR3(BVSD)=O.528,PWesternIntegrinα9(BVSC)=0.914,PIFIntegrinα9(BVSC)=O.593).(3)Compared with that of no inhibitors, the quantity of cell mig ration suffered a modest decrease with blocking VEGFR3 and Integrinα9(P=0.000), and the effect on cell migration quantity had no statistical difference between the two inhibitors at the concentration above(P=0.048﹥0.05),while the numbers of cell migration was further reduced wh en blocking VEGFR3 and Integrinα9 at the same time(P=0.000).ConclusionThere are two independent pathway :VEGF-C/VEGFR3 and VEGF-C/Integrinα9, in the differentiation process from stem cells into LECs;VEGFR3 and Integrinα9 play major role in the migration of induced c ells.