| Cardiovascular disease has been global disease with high morbidity and mortality. The morbidity and mortality of coronary heart disease (CHD) have been rising due to the improvement of people’s living standard. Acute myocardial infarction (AMI) is the main factor in causing death. The lost cardiomyocytes cannot be replenished by the regeneration, but only by replacement with the scar tissue, which finally results in the ventricular remodeling, cardiac systolic dysfunction and chronic congestive heart failure(CCHF).The ideal repair method on morphology and function is restoration by cells with high differentiation potential. Bone marrow mesenchymal stem cells (BMSCs) become the hot study of donor cells in the tissue engineering because of its ease to obtain, the strongly proliferative ability, the extensive proliferation in vitro, and the multilineage differentiation potential. In view of the fact that the state of injectioned seed cells is the key to the treatment, selection of inducing agent is very important. Common inducing agent consists of chemical drugs, various cytokines and traditional Chinese medicine effective component. Physical interventions such as electrical stimulation, magnetic pre intervention and the role of mechanical forces have also been mentioned. Biological factors such as simulation of myocardial microenvironment, tissue lysate co-culture and related gene are beginning to appear. And some are combined to be used in the process of culture. Bone morphogenetic protein-2(BMP-2) and Salvianolic acid B(Sal-B) are used as the inducers for BMSCs to differentiated into myocardial cells in vitro in this study. The morphological changes of the cells and the expressions of specific genes were observed during the differentiation. And the combined application of the two inducers was compared with the separate application to find a better induction method with high myocardial differentiation rate, and to provide an experimental basis for the clinical application of BMSCs in myocardial function recovery.The rat BMSCs were isolated, cultured in vitro and identified. BMSCs were isolated from the limbs long bone marrow of three weeks SD rats by the method of whole bone marrow wall-attaching cultivation. The shapes of BMSCs were observed under a phase contrast microscope. Primary cultured cells adhered partially in12h and began to change their appearance in24h. Part of these cells shapes stretched, volume increased and attached to the wall of culture dish. Part of cells stretched out protuberance which were blunt but smooth in72h. When cells with big and full nuclears were spindle-shaped, diamond-shaped or polygonal, they looked like fibroblast. After passage small and circular hematopoietic stem cells which were not adhered to the wall decreased gradually. There were more polygonal and spindle cells in the field of vision. The shapes were consistent, aligned, grew faster, and required a shorter generation time.The isolated cells were identified by immunocytochemical method. The results showed that CD29(fibers connecting receptor) was positive stain and CD34(hematopoietic stem cell marker) was negative stain in the isolated BMSCs, showing that these isolated cells expressed stromal cell antigen, not white cell antigen. It consistented with mesenchymal stem cell characteristics.The trace of the growth curve of the rat BMSCs showed that the separated BMSCs were not proliferated in the early24hours and grew slowly on the second and the third day. They entered the logarithmic growth phase and grew quickly on the fourth day. Then cell proliferation rate slowed down and they entered the plateau phase on the seventh day. The growth patterns were similar in every generation of the cells. After cryopreservation and resuscitation operation, the growth patterns of frowzen cells did not change. The cell viability was not affected. The generation time was similar to that before the cryopreservation and resuscitation operation.The experimental subjects were the second generation rat BMSCs which grew well and had homogeneous forms. They were divided into four groups after48h, and the induction culture medium with BMP-2, Sal-B and BMP-2+Sal-B were added in1st,2nd,3rd groups and the4th group was blank control. The induction culture medium was changed to common culture medium after3days and conventional culture for28days. Whether there were myocardial cells in the induced rat BMSCs or not could be found with morphological and molecular level test.Microscopically:cells of each group adhered to the wall, stretched, and extended out protrusions after48h induction culture. Cells appeared in polygonal, spindle and other forms. Cell shapes became more uniform gradually and appeared long fusiform in shape. And Orientation of cell was consistent1week later. Cells connected with each other and orientation also appeared obvious direction with part of them arranged in whorls4weeks later. Specimens were prepared for testing.Cell shapes were observed with hematoxylin and eosin(HE) stain. Streptavidin/peroxidase(SP) method immunocytochemical stain was used for identification of cardiomyocyte surface markers desmin、α-sarcomeric actin、cardiac troponin T(cTnT)、cardiac troponin I(cTnI) and P38-MAPK. The results showed that desmin、α-sarcomeric actin、cTnT、cTnI、 P38-MAPK were all positively expressed in the induced BMSCs in each group. On the other hand, desmin、α-sarcomeric actin、 cTnT、cTnI、 P38-MAPK were negative in the blank control group. Immunofluorescence stain was used for observation of coexpression on a-sarcomeric actin and cTnT markers in cells. The differentiated cells appeared to be the single or group distribution. Cells were spindle-shaped or branched, with one or two round nucleus located in the center. Part of the cells expressed only a-sarcomeric actin, while part of the cells expressed cTnT. Most of the cells expressed both surface markers.Transmission electron microscope(TEM) showed that, induced cells had abundant organelles, with an oval nucleus located in the center of the cells. These organelles contained a large number of rough endoplasmic reticulum, mitochondria, glycogen and ribosomes. Myofilament disposed parallel in the cytoplasm, with light and dark transverse striation. It showed a characteristic of myofilament installation during its development.Expressions in mRNA levels of GATA-4, a-MHC and Nkx2.5were detected by semi-quantitative RT-PCR. The results showed that, the GATA4and Nkx2.5genes expressed weakly at7d, enhanced at14d and decreased at28d; a-MHC gene was not expressed at7d, expressed weakly at14d and enhanced at28d. Gene expression of combined induced group was stronger than that of other groups at each time point significantly.It can be seen that in this experiment:1Rat BMSCs had isolated with whole bone marrow wall-attaching cultivation in vitro can be induced and differentiated as the "seed cells" for tissue engineering.2Cultured rat BMSCs can be induced by BMP-2, Sal-B and the two union into cardiomyocytes in vitro.3The application of the combined induction of BMP-2and Sal-B had a better effect than that of the separate induction in the differentiation from BMSCs to cardiomyocyte. |
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