The Role Of CD73 In The Myocardial Differentiation And The Secretion Of VEGF?TNF-? Of Adipose-derived Mesenchymal Stem Cells

BackgroundAdipose-derived mesenchymal stem cells(ADMSCs)has become one of the most promising stem cells for the cardiomyocytes transplantation and tissue engineering,ADMSCs are composed of different subgroups which has different biological characteristics and differentiation potential.The present study is aimed at the mixed ADMSCs,lacking the pertinence and effectiveness,it may also be one of the important reason for the limited effects of cell therapy.Therefore,it is necessary to find the prepotent subgroup of myocardial differentiation and study its biological characteristics,which can help to improve the relevance and effectiveness of cell therapy.ObjectivesExplore the role of CD73 in the myocardial differentiation and cytokine secretion of adipose-derived mesenchymal stem cells,proving CD73 molecule in the repair of myocardial function.Methods1.Mouse adipose-derived mesenchymal stem cells(ADMSCs)were culcured and indentified in vitro.Detect the expression of surface markers CD29?CD44?CD73?CD45,and the potential of differentiating into osteoblasts,adipocytes and cardiomyocytes.2.Select the subpopulation of CD73+and CD73-by flow cytometry.Inhibit the expression of CD73 of group CD73+-APCP on ADMSCs with its specific inhibitor adenosine 5?-?,?-methylene diphosphate(APCP),then divide into three groups:groupCD73+?group CD73-and group CD73+-APCP.3.Buid the lentiviral vector:CD73 over expression vector(CD73-OE),CD73 interference vector(CD73-RNAi)and null-vector-GFP(C-GFP),and then affect ADMSCs in vitro.Detect the transfection efficiency of lentiviral by immunofluorescence.4.Group CD73+?group CD73-and group CD73+-APCP ADMSCs were induced into cardiomyocytes in vitro.Detect the expression of cardiac troponin T for each group by flow cytometry and immunofluorescence,respectively.5.Collect the supernatant and detect the concentration of VEGF and TNF-? by ELISA.Results1.ADMSCs could differentiate to osteoclasts,adipocytes and cardiomyocytes-like cells in vitro.The result of flow cytometry showed ADMSCs were positive for CD29(53.1±1.1%),CD44(34.8±2.1%),while the expression of CD73 was unstable from 1.8%to 19.7%.ADMSCs were negative for CD45.2.After being induced 21 d by 5-aza,the result of immunofluorescence showed that all the groups could express myocardial specificity protein cTnT,the positive rate of cTnT:group CD73+85.0±6.3%,group CD73-2.1±0.6%,group CD73+-APCP 4.8±1.7%;the result of flow cytometry showed that CD73+ADMSCs 44.9%,CD73-ADMSCs 1.5%,group CD73+-APCP 3.3%.3.The concentration of VEGF:before being induced by 5-aza,group CD73+-APCP ADMSCs have dramatic decline(P<0.01)compare with group CD73+ADMSCs and group CD73-ADMSCs.At d10 after being induced by 5-aza,group CD73+-APCP ADMSCs have dramatic decline(P < 0.01)compare with group CD73+ADMSCs and group CD73-ADMSCs,group CD73+ADMSCs have obvious enhancement(P<0.05)compare with group CD73-ADMSCs.At d20 after being induced by 5-aza,group CD73+-APCP ADMSCs have dramatic decline(P<0.01)compare with group CD73+ADMSCs and group CD73-ADMSCs,group CD73+ADMSCs have obvious enhancement(P < 0.05)comparewith group CD73-ADMSCs.The concentration of TNF-?:group CD73+-APCP ADMSCs have dramatic decline(P < 0.01)compare with group CD73+ADMSCs and group CD73-ADMSCs,while group CD73-ADMSCs compared with group CD73+ADMSCs,there was no statistical significance.4.After ADMSCs were affacted by lentiviral,the concentration of VEGF:group CD73-OE was higher(P<0.05)compare with group CD73-RNAi and C-GFP;The concentration of TNF-?:group CD73-OE was lower(P < 0.05)compare with group CD73-RNAi and C-GFP.ConclusionsCD73 could promote ADMSCs differentiate into cardiomyocytes-like cells and promote the secretion of VEGF,inhibit the secretion of TNF alpha.