| Cardiovascular disease has been global disease with high morbidity and mortality. Due to the improvement of people’s living standard, morbidity and mortality of coronary heart disease continue to rise. Acute myocardial infarction (AMI) become an important cause of death. The number of the cells is reduced of myocardial cell necrosis after the occurrence of acute myocardial infarction. Myocardial cells can not be regenerat, so necrotic myocardium is replaced by scar tissue. Finally, it result in a decline in systolic function and congestive heart failure. So myocardial cell angioplasty, that is, injection of cells with differentiation capacity to colonize to the lesion site and differentiation for functional heart cells to repair the damage become a popular direction of the current study.After the effort of researchers in recent years, bone marrow stromal mesenchymal stem cells (BMSCs) which comes from their own have been found with the following advantages:1,They are easy to obtain, and have the genetic stability and portability for long-term culture in vitro.2, Their proliferation ability is strong and can proliferate extensively in vitro.3, They have multiplex differentiation potential and can differentiate into bone, cartilage, fat, liver, neural, endothelial, smooth muscle, skeletal muscle cells and cardiomyocytes in the appropriate microenvironment.4, They are easy to be implanted with exogenous gene which can be integrated into the genome of BMSCs and express stably without affecting their stem cell characteristics. And the expression of exogenous gene influenced by the microenvironment with tissue specificity.5, The application of them has no restriction of medical ethics and immune rejection. So BMSCs become the ideal target cells in gene therapy and are the most popular donor cells in the study treatment of cardiovascular diseases.In view of this, this study have selected Salvianolic acid B and tanshio IIA as inducers inducing BMSCs differentiated into myocardial cells in vitro. During differentiation, observe the morphological changes of the cells as well as comparing the combined application of two inducers with the separate application so as to find out which is more favorable for cell differentiation. Then find a better induction method, improve myocardial differentiation rate, provide experimental basis for the clinical application of BMSCs in myocardial function recovery.BMSCs were isolated from the limbs long bone marrow of2weeks 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 (100μg/mL)/streptomycin (100μg/mL). Change half of the nutrient medium12h after inoculation and all after24h, then one time every other day. The morphology of BMSCs was observed under a phase contrast microscope.The cells were identified by immunocytochemical method of cell surface markers in for to determination. Select the fourth generation cells which are grows well, with uniform ed shape and90%confluence and prepare cell slide. Fix them with40g/L paraformaldehyde. Immunocytochemical stain for identification of cell surface markers CD29and CD34. Trace the growth curve of the rat BMSCs in order to understand its growth characteristics. Select the second, fourth and sixth generation cells which are grows well, uniform shape and90%confluence inoculate at24holes plate with a1×107/L density. Digest3holes of cells, count and draw diagram every24h.Experimental object is fourth generation rat BMSCs which were grew well and had homogeneous forms. Divide them into four groups after48h. Add in induction culture medium with salvianolic acid B, tanshio IIA and Sal-B+tanshio IIA in1,2,3groups and common culture medium in blank control group. Change induction culture medium into common culture medium after3days and have conventional culture for28days. The morphological and molecular level test will find that whethre there are myocardial cells in the induced rat BMSCs or not. Select the28days induced cells which were grew well and uniform shape prepare cell slide. Fix them with40g/L paraformaldehyde. SP method immunocytochemical stain for identification of cardiomyocyte surface markers desmin, a-sarcomeric actin, cTnT and Cx43. Obsene of coexpression about desmin and cTnT markers in cells with Immunofluorescence stainPrimary cultured cells were adhereed partially in12h and began to change their appearance in24h. Part of these cells were morphology stretching, volume increase and attached to the wall of culture dish. Part of cells were stretched out protuberance which were blunt and smooth in72h. When cells with big and full nuclear were spindle-shaped, diamond-shaped or polygonal, they look like the fibroblast. Cells covered the wall of culture dish after1week. And the volume increase more, with more varied form.The results showed that the CD29(fibers connecting receptor) was positive stain and CD34(hematopoietic stem cell marker) was negative stain in the isolated BMSCs. The expression of these isolated cells with stromal cell antigen and without white cell antigen consistent with mesenchymal stem cell characteristics.We can see in the diagram that the separated BMSCs did not proliferate in the early24hours and they grew slowly in the second and the third day. They were entered the logarithmic growth phase and grew quickly in the fourth day. Then cell proliferation rate-of them slow down and they were entered the plateau phase in the seventh day. Growth pattern were similar in every generations of cells.Microscopically:cells of each group were adhered to the wall, stretch, and extend out protrusions after48h induction culture. Cells were appeared polygonal, spindle and other forms. Cell morphology became more uniform gradually and appears long fusiform in shape. And Orientation of cell was consistent1week later. Cells were connected with each other and orientation also appeared obvious direction with part of them arranged in whorls4weeks later.The results showed that desmin、a-sarcomeric actin、cTnT and Cx43were all positive expression in the induced BMSCs in each group. On the other hand, desmin、a-sarcomeric actin、cTnT and Cx43were negative in the blank control group. Cells were spindle-shaped or branch, with one or two round nucleus located in the center. Part of cells were expressed only the desmin, while part of cTnT. Most of the cells were expressed both surface markers.1Rat BMSCs which were isolated with whole bone marrow wall-attaching cultivation in vitro have a strong proliferation and differentiation ability. They had showed a growth law similar with stem cell characteristics. They were expressed CD29(fibers connecting receptor) but not CD34(hematopoietic stem cell marker). Their proliferative capacity was not affected after cryopreservation and anabiosis operation. So BMSCs can be induced differentiation as the "seed cells" for tissue engineering.2Cultured rat BMSCs can be induced by salvianolic acid B, tanshio IIA and the two union into cardiomyocytes in vitro. The morphological observation, immunocytochemical detection result showed that, the myocardial-differentiation BMSCs had early myocardial cell structure and markers expression. It suggested that they may participate in the transcription regulation in the differentiation from BMSCs to cardiomyocyte.3Differentiation rate coming from immunocytochemical stain results were suggested that the application of salvianolic acid B and tanshio IIA combined induction surpasses using each of them separately obviously in the differentiation from BMSCs to cardiomyocyte. |
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