The Impact On Migration Ability Of The Tip Endothelial Cell By The PI3K/Akt Signal Pathway Mediated By SDF-1/CXCR4 Axis

Part ?Isolation and differentiation of human endothelial progenitor cells,vascular endothelial cells and Tip vascular endothelial cells Objective:To investigate the methods of isolation and cultivation of human endothelial progenitor cells(EPCs).To research the culture conditions of human endothelial progenitor cells in vitro and biological characteristics and confirm the peripheral blood is the another ideal source of human endothelial progenitor cells except the bone marrow.The human endothelial progenitor cells were differentiated into the vascular endothelial cells and Tip vascular endothelial cells.This can provide the cells for next experiment and a reliable basis for mechanism of stem cell-derived angiogenesis.Methods:Peripheral blood mononuclear cells were isolated by density-gradient centrifugation and cultured in endothelial growth medium-2 in the presence of vascular endothelial growth factor and renew the culture solution every 3 to 4 days.Observe the morphology of the cells every day by microscopy.After 21 days,collect the adherent cells and identify the endothelial-specific markers PDGF-B and D114 of Tip endothelial cells by flow cytometry,reverse transcriptase-polymerase chain reaction(RT-PCR)and Western Blot.Results:Most of the cells were adherent growth after 4 days' culture.Most were able to form fusiform endothelial cells and some gather clouds to form a clonal colony after 7 days' culture.After 21 days,the pebble-like cells spread the filopodia and connect each other.Flow cytometry,RT-PCR and WB demonstrated the presence of PDGF-B and D114 mRNA.Conclusion:Endothelial progenitor cells could be successfully isolated and cultured from perpheral blood mononuclear cells.The human endothelial progenitor cells can be differentiated into the vascular endothelial cells and Tip vascular endothelial cells.Part ?Transwell migration experiment of the Tip endothelial cellAbjective:To study the biological impact of PI3K/Akt signal pathway on the Tip endothelial cell,and prove that PI3K/Akt signal pathway is the downstream mechanism to regulate the migration of Tip endothelial cell.This experiment was aimed to observe the impact of SDF-1/CXCR4 axis and PI3K/Akt signal pathway on the migration ability of Tip endothelial cell,which was cultured in vivo,by using a certain concentration of stromal cell derived factor-1(SDF-1),CXCR4 receptor inhibitor AMD3100 and PI3K blockers LY294002 to lay the foundation for clinical use of Tip endothelial cell transplantation for therapeutic angiogenesis.Methods:Collect Tip endothelial cells cultured for 21 days in the first part of the experiment,and ues pretreatment of(serum-free EGM balanced in incubator for 1 hour)Transwell Chambers(12 holes/plate,aperture 8 u m)to make experiments.Cell suspension was divided into seven groups:(1)negative control group:add 100ul cell suspension in the upper room,and add serum-free EGM without SDF-1 in the down room;(2)AMD3100 control group:add 100ul cell suspension in the upper room with pretreated CXCR4 inhibitors AMD3100,and add serum-free EGM without SDF-1 in the down room;(3)LY294002 control group:add 100ul cell suspension in the upper room with pretreated PI3K inhibitors LY294002,and add serum-free EGM without SDF-1 in the down room;(4)experimental group:add 100ul cell suspension in the upper room,and add serum-free EGM with SDF-1(100 ng/ml)in the down room;(5)CXCR4 blocking group:add 100ul cell suspension with pretreated CXCR4 inhibitors AMD3100 in the upper room,and add serum-free EGM with SDF-1(100 ng/ml)in the down room;(6)LY294002 block group:add 100ul cell suspension with pretreated PI3K inhibitors LY294002 in the upper room,and add serum-free EGM with SDF-1(100 ng/ml)in the down room;(7)exogenous PIP3 group:add 100ul cell suspension with pretreated CXCR4 inhibitors AMD3100 and PI3K phosphorylation PIP3 in the upper room,and add serum-free EGM with SDF-1(100 ng/ml)in the down room.Transwell experiment detected the migration ability of Tip endothelial cell influenced by SDF-1/CXCR4 axis and PI3K/Akt signal pathway and intervened by AMD3100 and LY294002.Results:The migration ability of Tip endothelial cells were different from each other in 7 groups(F =610.25,P<0.05).Compared with negative control group,the number of migration cells in AMD3100 control group and LY294002 control group had no significant difference(t=0.421,P>0.05).Compared with negative control group,the number of migration cells increased in experimental group(t=53.241,P<0.05).Comp-ared with the experimental group,the number of migration cells in CXCR4 blocking group and LY294002 block group were less(t=50.715 and 38.300,P<0.05).When putting exogenous PIP3 into CXCR4 blocking group,the chemotaxis of SDF-1 can be observed again and the number of migration cells has no significant difference compared with the experimental group(t=0.631,P>0.05).And the number of migration cells in exogenous PIP3 group were more than those in CXCR4 blocking group(t=50.084,P<0.05).Conclusion:SDF-1/CXCR4 axis and PI3K/Akt signal pathway take part in the migration of Tip endothelia cells and PI3K/Akt signal pathway is the downstream mechanism of Tip endothelia cells migration mediated by SDF-1/CXCR4 axis.