| Ischemic heart disease induced by atherosclerosis easily causes acute myocardial infarction, which is a frequently occurring disease harmful to the people’s health. Cellular cardiomyoplasty can realize the structure repair and restore founction of cardiocytes from the root, and is one of the promising treatment methods to improve the cardic founction of heart attack patients. As tissue engineering seed cells, the international focus in resent years the bone marrow mesenchymal stem cells (BMSCs) have incomparable advantages. BMSCs showed a good prospect of medical treatment because of convenience to get, none immune, proliferation ability, and multi-directional differentiation potential capacity.This study selects dimethylsulfoxide(DMSO) as an inducer induced BMSCs differentiated into myocardial cells in vitro. During differentiation, observe the morphological changes of the cells and the expression of specific gene. Then find a better induction method, improve myocardial differentiation rate, provide experimental basis for the clinical application of BMSCs in myocardial function recovery.1BMSCs were isolated from the limbs long bone marrow of3weeks ages SD rats with whole bone marrow wall-attaching cultivation. Culture them at37℃in humid air with5%CO2in iscove’s modified dulbecco’s medium (IMDM),10%fine fetal bovine serum (FBS), penicillin (100mg/L)/streptomycin (100g/L). Change half of the nutrient medium12h after inoculation and change all after24h, then one time again every2days. The morphology of BMSCs is observed under a phase contrast microscope. The2nd-generation BMSCs were induced by0.6%.0.8%、1.0%DMSO for72 h,(group A, B and C respectively), and common culture medium in blank control group(group D). Change induction culture medium into common culture medium after3days and conventional culture4weeks.2Immunocytochemical staining:SP method immunocytochemical stain for identification of cardiomyocyte surface markers desmin, a-sarcomeric actin, cTnT, cTnI and P38-MAPK.3Immunofluorescence staining:Immunofluorescence stain for observation of coexpression about desmin and cTnT markers in cells of group C.4Select the28days induced cells of group C which grew well and uniform shape prepare electron microscope specimen. The ultrastructure of induced cells were observed by transmission electron microscope.5Primary cultured cells adhere partially in24h. Part of these cells morphology stretching, volume increase and attached to the wall of culture dish in72h. When cells with big and full nuclear are spindle-shaped, diamond-shaped or polygonal after1week. And the volume increase more, with more varied form. The morphology of BMSCs were different in each treatment group. BMSCs in A, B group on7d were the same as the control group. BMSCs in C group on28d showed fusiformshape, orientating with one accord, forming myotubule-like structure.6Immunocytochemical staining:BMSCs induced by different concentrations of DMSO on28d could be identified by the positive staining for desmin, a-actin, cTnT, cTnI and P38MAPK. The positive rates were all higher than that of the control group. The positive rates of desmin, α-actin, cTnT, cTnI and P38MAPK in C group were all greater than that of A, B group (P<0.05).7Immunofluorescence staining was showed that bright red fluorescence in the cytoplasm could be observed in the desmin-positive cells, green fluorescence in the cytoplasm could be observed in the cTnT-positive cells while the part that overlaps were yellow. 8Transmission electron microscope shows that BMSCs have abundant and developed organelles, with an oval nucleus located in the center of the cells. These organelles contain large number of rough endoplasmic reticulum, mitochondria, glycogen and ribosomes. Myofilament disposes parallel in the cytoplasm.DMSO may induce BMSCs to acquire cardiogenic phenotype by morphology and immunofluorescence staining and1.0%DMSO may be a suitable induced concentration.DMSO may induce BMSCs to acquire cardiogenic phenotype by immunofluorescence staining and transmission electron microscope. |
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