The Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Vascular Endothelial Cells In Vitro

[Objective] :To investigate the feasibility of differentiation of bone marrow mesenchymal stem cells(MSCs) into vascular endothelial cells in vitro,and compare the effects of ATRA (all trans — retinoic acid) and IGF —1 (insulin-like growth factor— 1) on cell differentiation.[ Methods ] :MSCs were isolated and purifided by density gradient centrifugation and adherent culture.After 3-5 generations according to our method,the cultured typical MSCs were reseeded onto culture medium I(M199 medium containing + 10%fetal bovine serum + VEGF10ng/ml+bFGF5ng/ml +IGF-1 2ng/ml)and culture medium A(M199 medium containing +10%fetal bovine serum+VEGF10ng/ml+bFGF5ng/ml +ATRA1 μ mol/L) as experimental group I and A.MSCs cultured without any special growth factor were labelled as negative group M, and endothelial cells were treated as positive group E.Changes of morphology and numbers of cells were observed under light microscope.and electron microscope.Theexpressions of factor VDI s CD34 -. KDR > vWF were detected by immunohistochemical staining -. flow cytometry , RT—PCR and Western —blotting respectively.[Results] :Some cells of group I and A turned from spindle (the same shape of group M) into irreugar or round(just like the cells of group E).Some cells of group I and A died and the mean cell density was lower than that of group M.Though the speed of cells proliferation of group A was slower than that of group I,it became faster from the forth day.Immunohistochemical staining of factor VDI showed 7.57 + 4.21% cells of group I and 8.10 + 3.73% cells of group A were positive cells.(p>0.05).There were no significant difference between these two groups.Weibel—Palade articles can be found in the cytoplasm of some cells from group I and group A by transmission electron microscope.FCM(flow cytometry) analysis showed that the fluorescence intensity of KDR, CD34 expressed in cells of group I and A was significantly higher than that in group M.KDR mRNA can be found positive in groupL A and E by RT— PCR,while negative in group M.Western—blotting assay showed that vWF was expressed in cells of groupL A and E,and can not be found in group M.[Conclusion] : MSCs can be isolated and purifided by density gradient centrifugation and adherent culture; A proportion of MSCs can be induced to vascular endothelial cells both in culture medium I(VEGF +bFGF +IGF-1) and culture medium A(VEGF +bFGF +ATRA); Culture A showed lowerability of inducement in the beginning,and higher ability gradually.