Migration And Proliferation Of C-kit~+ Cardiac Stem Cells In Accelerating Neonatal Mice With SDF-1α In Vitro

BackgroundStem cells have been used in clinical and basic research of myocardial infarction. In recent years, the research and discovery of cardiac stem cell have opened a new way in the therapy of myocardial infarction. Once the myocardial necrosis after injury, they cannot produce promptly and efficiently large amounts of parenchymal cells. Fibroblast proliferation rapidly and secrete matrix, lead to the formation of fibrous scar, which limit the damage of myocardial tissue regeneration and prevent cardiac stem cell migration to the damage of necrotic area. Therefore, how to activate cardiac stem cells in situ quickly, promote stem cell proliferation, migration and homing, explore the therapy of myocardial infarction molecular target become the research focus in the field.ObjectiveTo probe the migration and proliferation of c-kit+ cardiac stem cells in accelerating neonatal mice with Stromal cell-derived factor-1α in vitroMaterials and methodsThe SDF-1α gene sequence was retrieved from GenBank, and the primer sequence were designed. The virus construction protocol was submitted to the biology company to build overexpression lentivirus vector with SDF-1α.The cardiac fibroblasts were primary cultivated with the enzyme digestion method, and the cardiac fibroblasts were transfected SDF-1α overexpression lentivirus (LV-SDF-1α) in different concentration to detect the best tranfection concentration. The expression of SDF-1α were detected with Dot-blot method in the liquid supernatant cultivated by the cardiac fibroblasts transfected LV-SDF-1α in different time. The cultural liquid supernatant of cardiac fibroblasts transfected the non-carrier virus (CON 145) as the control group.The primary heart cells were cultivated with the tissue block culture method till the 20th day. The c-kit+ cardiac stem cells were obtained with the flow cytometry (FCM) sorting, and were continuously cultivated.The morphological change of c-kit+ cardiac stem cells were observed with phase microscope. The expression of the early-stage marker of the cardiac muscle (Nkx2.5) and the cardiac muscle cell specificity marker cardiac troponin T (cTnT) were detected with the double-labeling immunofluorescence technique in differentiated c-kit+ cardiac stem cells.The c-kit+ cardiac stem cells and the fibroblasts transfected the lentivirus (non-carrier virus and LV-SDF-1α) were cocultivated, and the migration of c-kit+ cardiac stem cells were researched with Trans well chamber. The SDF-1α expression quantity were carried out with the enzyme-linked immunosorbent assay kit in the experimental group and the control group.The proliferation of c-kit+ cardiac stem cells were detected with Click-iT(?)EdU cell proliferation assay kit.Measurement data showed with mean±tandard deviation. Groups were compared using ANOVA, and LSD analysis was used to compare the group with SPSS 17.0 software for statistical analysis. P<0.05 was considered statistically significant.ResultsThe cardiac fibroblasts transfected LV-SDF-1α displayed GFP green fluorescence expression in 1×107 TU/ml and 1×108 TU/ml dose. The SDF-1α expressing of cardiac fibroblasts transfected LV-SDF-1α in the liquid supernatant showed the most amount in the 4th day after transfection with Dot-blot method.The positive rate of c-kit+ cardiac stem cells showed about 40% with flow cytometry sorting. The c-kit+ cardiac stem cells showed the morphological change from small round cells to long spindle or irregular oval shape cells for continuously cultivating about 20 days, which displayed cTnT positive expression with immunofluorescence technology.The result of cocultivating the c-kit+ cardiac stem cells and cardiac fibroblasts for transfected SDF-la overexpression lentiviral vector through Trans well technique presents that, the expression of SDF-la had considered statistically significant between groups (P<0.05), and the migration amount of the c-kit+ cardiac stem cells were obviously increased compared with the control group of transfection non-carrier or ADM3100 group (P<0.05).The result of Edu traces showed that the proliferation of c-kit+cardiac stem cells did not have obvious difference comparing with that of the control group.ConclusionThe fibroblast transfected lentiviral vector (LV-SDF-1α) can highly express (secrete) SDF-1α, and SDF-la can accelerate the migration of c-kit+cardiac stem cells, and cannot promote the proliferation of c-kit+ cardiac stem cells in vitro.