The Study Of Mechanisms Of The GIT1 Effects The Differentiation Of Bone Marrow-derived Mesenchymal Stem Cells To Endothelial Cells

Objective: It is well established that GIT1 effects the angiopoiesis of lung and fracture healing of mice.The GIT1 knock out mice had bad lung development and fracture healing,but the mechanism is not well understood.The current research focused on the differential of differentiation between wild type and GIT1 knock out mice.Methods: GIT1 heterozygous mice bred by families,then we identified the type of the second generation by PCR to get homozygote mice.MSCs were isolated from the bone marrow of mice.The GIT1 wild type and knockout MSCs cells were isolated and cultured,which were divided into two groups respectively at passage 2.One group was study group treated by VEGF-165(50 ng/ml)in vitro and the other was control group cultured in L-DMEM.Then there were four groups,wild type control group(group A),wild type study group(group A1),GIT1 knockout control group(group B)and GIT1 knockout study group(group B1).The protein expression of VEGFR-2,VEGFR-3 and p VEGFR-2,p VEGFR-3 were detected by western blot and the EC markers(v WF,PECAM-1 and VE-Cadherin)were compared by flow cytometry.Passage 2 BMSCs of wild type mice were taken,and 4 groups were divided according to the different culture medium.The cells cultured in L-DMEM named as GroupⅠ;the cells cultured in the media added SAR131675 which selectively blocked VEGFR-3 named as group Ⅱ;the cells treated by VEGF-165 named as group Ⅲ;the cells treated by VEGF-165 and SAR131675 named as group Ⅳ.The EC markers(v WF,PECAM-1 and VE-Cadherin)were compared by flow cytometry.Results: The western blot results showed that the protein expression of group A1 was higher than group A,group B,group B1.The flow cytometry results showed that the group A1 had a significant increase of the EC markers v WF,PECAM-1 and VE-Cadherin than group A,group B,group B1(P<0.05)and group B1 was significantly higher than group A,group B.There were no significant different between group A and group B(P>0.05).In the VEGFR-3 blocked experiment,the flow cytometry results showed that he group Ⅲ had a significant increase of the EC markers v WF,PECAM-1 and VE-Cadherin than group Ⅰ,group Ⅱ,group Ⅳand group Ⅳ was significantly higher than group Ⅰ,group Ⅱ(P<0.05).There were no significant different between group Ⅰ and group Ⅱ(P>0.05).Conclusion:The present study revealed that GIT1 mediates MSC differentiation into ECs via VEGFR-3.