Extracorporeal Simulate Oxidative Stress Of Acute Myocardial Infarction To Demonstrate The Effect Of Tenascin-C On Proliferation,Migration And Differentiation Of Mice Bone Marrow Mesenchymal Stem Cells

Background:A fair mount of animal experiments and clinical trials confirm that it’s safe and effective for stem cells to treat acute myocardial infarction by transplantation.Bone marrow stem cells has been the feasible stem cell for clinical therapy because of its convenient extracting and easy proliferation.Whereas,there is a bottleneck of stem cell therapy for its low rate of homing and proliferation after transplantation.Tenascin-C is a large hexameric extracellular matrix glycoprotein which can accelerate the development of heart during embryo development stage,but vanish in the normal adult myocardium.However,tenascin-c can expression in intermediate zone between infarcted myocardium and normal myocardium to participate in tissue repair and myocardial fibrosis after myocardial infarction.Toll Like Receptor 4 is a transmembrane protein which plays a key role in immunoreaction.Recent studies found that TN-C is a surface-active ligand of TLR-4 and can activate innate immunity and adaptive immunity when combined to TLR-4.BMSCs can express functionality Toll like receptor-4 which play a role in proliferation and migration through Mitogen-activated protein kinase,AKT,Wnt signaling pathway if be activated.Our team has proved that TN-C can facilitate magration of BMSCs,but can not affect its proliferation and differentiation.Hower,it is still unclear whether TN-C has the effect on migration,proliferation and differentiation of MSCs and whether TN-C exerts effect via combining to TLR-4 which is on the cytomembrane of MSCs.Objective:In the microeanvironment that extracorporeal hydrogen peroxide simulates oxidative stress of acute myocardial infarction,we investigate the effect of different concentration of tenascin-c on survival,migration and differentiated for bone marrow stem cells.And certify that whether tenascin-c exert biological effect via bonding to TLR-4 expressed on the cytomembrane of bone marrow stem cells and then activate MAPK,AKT,Wnt signaling pathway.Methods:3-4 generation BMSCs comes from C57/BL type mouse is used as experimental subject.Firstly,in the microenvironment that extracorporeal Hydrogen Peroxide simulates oxidative stress of acute myocardial infarction,CCK-8,Flow Cytometry,Transwell and ICC/IF is used to detect the effect of different concentration of TN-C on survival,migration and differentiation of BMSCs in the situation of different concentration of H2O2.Further,above-mentioned BMSCs is used to detect whether TN-C exerts its effect through bonding to TLR-4 on the surface of MSCs and its special molecular mechanism.In the microenvironment that extracorporeal Hydrogen Peroxide simulates oxidative stress of acute myocardial infarction and added in TAK-242 which is the specificity receptor of TLR-4,CCK-8,Transwell and Western Blot is used to discuss the effect of optimum concentration of TN-C on migration,proliferation and differentiation of BMSCs and on downstream signalling pathway.Results:The results of extracorporeal cultivation BMSCs with H2O2 show that 60-90umol/ml H2O2 can cause apoptosis of BMSCs,and low concentration of TN-C(1-50ug/ml)has no effect on apoptosis of BMSCs caused by H2O2.But high concentration of TN-C(100ug/ml)can reduce the number of apoptosis of BMSCs,scilicet can protect BMSCs from apoptosis.The Transwell show that H2O260-90umol/ml can promote migartion of BMSCs alone.H2O2 60-90umol/ml and 10-100ug/ml TN-C also can promote migration of BMSCs,and the rate of migration of BMSCs is better than cultured with H2O2 alone.Thus we can draw a conclusion that the optimum concentration of TN-C to promote migration and protection of BMSCs is 100ug/ml,in the situation of 60-90umol/ml H2O2.Repeat above experiment with TAK-242 which is the typical receptor of TLR-4.The result shows that the effect of TN-C on migration of MSCs obviously reduce and the number of apoptosis of MSCs increase.TN-C can not promote proliferation and differentiation of MSCs in the microenvironment of myocardial infarction.Further analysis of its mechanism shows that the effect of 100ug/ml TN-C promoting migration and reducing apoptosis of MSCs is because of binding to TLR-4 expressed on the cytomembrane of MSCs then activating it and further mediating the intracellular signalling pathway of MAPK,AKT,Wnt.Conclusion:In the case that extracorporeal 60-90umol/ml H2O2 simulats the microenvirenment of myocardial infarction,l00ug/ml TN-C can promote migration and restrain apoptosis of BMSCs from 60-90umol/ml H2O2.And that biological effect is exerted through TN-C combine with TLR-4 expressed on the cytomembrane of BMSCs then active MAPK、AKT、Wnt signaling pathway.TN-C can not promote proliferation and differentiation of MSCs in vitro.