Comparative Study Of Three Methods Induce Bone Marrow Mesenchymalstem Cells Into Cardiomyocyte-like Cells

Objective:To separate,purificate and amplify rabbit bone marrow mesenchymal stem cells through density gradient centrifugation.Completed bone marrow mesenchymal stem cells to induce cardiomyocyte-like cells, and to compare differentiation of the three different inducers of induced mesenchymal stem cell into cardiomyocyte-like cells,filtr the best inducible program.Methods:In vitro cultured and amplify rabbit bone marrow mesenchymal stem cells in density gradient centrifugation,and observed the shape of cells through the microscope. The fourth passage cells were divided intoⅠ,Ⅱ,Ⅲ,Ⅳ.then groupⅡ,Ⅲis divided into three subgroups again (ⅡA,ⅡB,ⅡC,ⅢA,ⅢB,ⅢC).10μmol/L 5-azacytidine was added Group I to induce; Group II (0.02umol/L AngiotensinⅡA was added GroupⅡA to induce,0.2μmol/L Angiotensin II was added GroupⅡB to induce,2μmol/L Angiotensin II was added Group II c to induce); GroupⅢ(1μg/L BMP-2 was added Group IIIAto induce, 10μg/L BMP-2 was added GroupⅢB to induce, 100μg/L BMP-2 was added GroupⅢc to induce); GroupⅣwas set up as control group. Induced with the inducer of each group 24 hours. After induction, washing.Then continue culture cells for 28d after change medium. the phenotype of induced cells were confirmed by immunohistochemical staining,color after 28 days.Results:(1)The speed of amplify the rabbit bone marrow mesenchymal stem cells is fast by density gradient centrifugation, all of these are spindle cells.(2) immunohistochemical methods was adopted to test cardiac-specific Troponin T and connexin 43.cTnT and Cx-43 has shown positive express in induced group, control group don't express.(3)In groupⅡ,Ⅲ,the expression of cardiac-specific protein are increasing with induced density. Group II c (0.2975±0.0249) is more than groupⅠ(0.2625±0.0306),The difference was statistically significant (P <0.01).group I is more than groupⅢc (0.2088±0.0300),The difference was statistically significant (P<0.01).Conclusion:(1)The rabbit bone marrow mesenchymal stem cells can be effective separated,cultured and amplified by density gradient centrifugation,(2)Undergo 5-azacytidine, angiotensinⅡ,bone morphogenetic protein-2 induce, bone marrow mesenchymal stem cells can differentiate into cardiomyocyte-like cells.(3) AngiotensinⅡ'seffects is better than 5-azacytidine, 5-azacytidine is better than bone morphogenetic protein-2.