Effects Of Angiopoietin-1 On Adhesion, Vigour And Apoptosis Of Mesenchymal Stem Cells Through Integrins

Objective:To investigate whether human recombinant angiopoietin-1 can promote adhesion and survival and inhibit apoptosis of rat bone marrow MSC,then discuss its anti-apoptosis pathway of signal transduction preliminary.Methods:Combining density gradient centrifugation and adherence separation to separate,cultivate rat bone marrow MSC In vitro and identify its surface antigen by flow cytometry,detect the adhesion effect of Ang-1 on MSC via counting the number of cells sticking to 24-well flat-bottom-tissue culture plates at different times.Trypan blue staining method and MTT test are used to survey the vigor effect of Ang-1 on MSC under serum starvation.Also we apply methods of Hoechst staining and Annexin V/PI to investigate its influence after inducing apoptosis by taxinol. Approach Ang-l's anti-apotosis through examining the expression of phosphorylation of Akt,total Akt,Bcl-2 andβ-actin by western blotting,and combing integrin antibody and signal transduction blocker.Results:As combination of cell morphology,growth features and surface antigen detection by flow cytometry instruments are all in line with the characteristics of MSC,we separate,culture and amplificat it successfully in vitro.Cell adhesion assay shows Ang-1 can promote the adhesion of MSC(P＜0.01 all ) at lday,2 days and 3days,which can be inhibited by EDTA,subunit of integrinβ1 antibody and RGD peptides(P＜0.01 all ).Ang-1 promotes MSC survival(1d:P＜0.01;2d:P＜0.005 ) and proliferation(P＜0.01 )under serum starvation,the same time protest MSC from apoptosis induced by taxinol(P＜0.01),RGD peptides can inhibit the Ang-l's above-mentioned actions.The expression of Ang-1 group's pAkt and Bcl-2 are increased,RGD peptides can inhibit two(P＜0.01).PI-3K/Akt transduction channel blockers Wortmannin can repress pAkt expression(P＜0.01) only,but not Bcl-2 (P=0.436) Conclusion:Highly purified MSC can be acquired through integrating density gradient centrifugation with adherence separation.Mediated via the Ligand on MSC's surface integrins,Ang-1 can promote the adhesion,survival and proliferation of MSC,and exert the anti-apoptosis effect by up-regulating Bcl-2.RGD peptides can restrain Ang-1's anti-apoptosis protective role.