| With the amelioration of people's living standards and alterations in diet, the incidence of cardiovascular disease had been ascending year after year. The short and long-term complications of myocardial infarction, in particular, not only threatened mankind's life, but also severely affected people's living quality. Despite the presence of human cardiomyocytes proliferation of the phenomenon had been reported in the papers, only 1% of cells updating rate was hard to make up for the loss of the cardiomyocyte cells therefore had little improvement on heart function. At present, the clinical treatment of myocardial infarction concentrated on the rescue of non-infarcted cardiomyocyte cells and the amendment of heart function, nevertheless, neither drug therapy, interventional treatment nor surgical procedures could restore the infarcted heart function thoroughly. The bereft cardiomyocytes would be replaced by the scar tissue, which led to cardiac systolic dysfunction and ultimately morph into heart failure(HF).The consummate therapeutic measures on myocardial infarction ought to achieve the effect of restoration morphology and function. Heart transplantation may, after all, be regarded as an effective modern treatment. However, because of lack of donor source, incapable of evading immunological rejection and costly surgery, It is difficult to generalize in clinical practice. In recent years, many scholars indicated that the cardiomyocyte-like cells differentiated from induced stem cells could displace infarcted cardiomyocytes cells and mend heart function, which, beyond doubt, have ushered flush of dawn to the treatment of ischemic heart disease. Bone marrow mesenchymal stem cells(BMMSCs) are regarded as a sort of pluripotent stem cells, and became a centralized research object on account of its convenient to acquire, liable to culture and proliferate in vitro, the potential of multilineage differentiation and autologous transplantation without immunological rejection. By far, it has become the ideal seed cells in the field of tissue engineering. Undoubtedly, the state of seed cells is the key to ensure the success of the transplantation treatment, in consequence, it is crucial to select inducing agents. Currently, major studies have been involved in different patterns of induction, such as drug induction, physical and biological factors intervention. Inducing agents contain chemicals, varieties of cytokines and Chinese medicine effective monomer. Physical interventions include biomimetic electrical stimulation, external magnetic field and the effect of fluid shear stress. Biological factors interventions, for example, the analog myocardial microenvironment in vitro, cell lysate cocultivation and related gene transfection. Previous researches had shown that some are combined to be used in the course of culture.Basic fibroblast growth factor pertains to the fibroblast growth factor family, which is widely distributed in almost all organs and tissues, most of them exists in the hypothalamus and pituitary. FGF-2 is a potent mitogen, which can stimulate the migration, proliferation and differentiation of various types of stem cells. Researches indicated that the viability and anti-apoptotic characteristics of MSCs transfected by FGF-2 were enhanced in hypoxic conditions. Wnt-11 belongs to the Wnt protein family, which is expressed in embryonic ventricle and atrium.Wnt-11 is essential to the normal development of cardiac function and controls the early development of myocardium. It has been pointed out that Wnt-11 governs the spatial organization of cardiomyocytes in the process of cardiomyogenesis. Some scholars believed that, in cell culture models, Wnt-11 signal transduction determined the fate of myocardial cells and promoted directed differentiation of cardiomyocytes. Based on the researches above, FGF-2 and Wnt-11 were applied to induce the differentiation of BMMSCs into cardiomyocyte-like cells in this project, with an aim to explore their function, observe the morphological changes of BMMSCs and analysis the expression of correlatively specific genes in the process of the differentiation. Meanwhile in terms of cell differentiation, to explore whether the effect of the combined inducers is better than that of the separate application or not. With a view to taking this opportunity to enhance the efficiency of cell differentiation and laying the foundation for clinical treatment of myocardial infarction by cell transplantation.The required bone marrow for the study was harvested from the long bone of three-week-old SD rats, using whole bone marrow culture method. The morphological characteristics of BMMSCs were observed with the inverted phase contrast microscope. Primary passage of BMMSCs had small cell nuclear and attached partially when cultured for 12 h, and then preliminary changes in cell morphology occurred in 24 h. part of the cell bodies spread, and clung to the growth plane of the culture flasks. protuberances with varying size and length stretched out from part of these cell bodies in 72 h, cells were mainly short shuttle-shaped, oval and irregularly-shaped ones were also visible. Which were similar with fibroblasts in appearance. After the passage hematopoietic stem cells which grew suspendedly could be thrown away gradually. The shapes of cells were arranged consistent with the same over time.Previous studies have shown that BMMSCs can express the surface antigens of mesenchymal cells and endothelial cell lines CD29, CD50, CD90, and do not express the surface antigens of hematopoietic cells CD14, CD45. BMMSCs were detected by flow cytometry for surface antigens. The consequences showed that the positive rate of CD29, CD45, CD90 was 97.9%, 0.4%, 99.5% respectively, revealing that these cells expressed stromal cell antigen, rather than hematopoietic stem cell antigen. It conformed with the characteristics of mesenchymal stem cells.The second generation rat BMMSCs which grew well were selected as the experimental subjects. When cultured for 48 h, BMMSCs were split into four groups, and the culture medium added with FGF-2, Wnt-11 and FGF-2+Wnt-11 were regarded as the induction group A, B and C, Meanwhile, group D was blank control. The culture medium added with inducing agents was replaced by complete medium after 72 h, while group D was only treated with complete medium and conventional culture for 4 weeks. The induced rat BMMSCs were detected at the morphological and molecular level.Observed morphological changes in the cells with the inverted phase contrast microscope: cells of each group clung to the wall, spread, and with blunt and smooth protuberances stretching out of the cell bodies after induction culture 48 h. Cells were mainly shuttle-shaped, oval and irregularly-shaped ones were also visible. The morphology and arrangement of cells gradually became uniform, long shuttle-shaped 1 week later. Connections between cells grew tightly, and cells orientation appeared evident direction, moreover, some of them arranged in swirls 4 weeks later.When BMMSCs were cultured for 4 weeks, immunocytochemical method was used to detect the expression of cardiomyocyte markers Desmin, cTnI and Connexin43 in each group. The consequences demonstrated that Desmin, cTnI and Connexin43 were all positively expressed in each induced group. Whereas the expression of them in the blank control group was slight positive or negative.Transmission electron microscope(TEM) revealed that, organelles such as a mass of rough endoplasmic reticulum, mitochondria, meanwhile, some glycogen and ribosomes were visible in the cytoplasm of these cells in each induced group. In addition, myofilament arranged in parallel in the cytoplasm. BMMSCs in induced groups had an oval nucleus which located in the center. These ultrastructural characteristics of BMMSCs in induced groups were similar to myocardial cells.Expressions of cardiac transcription factor GATA-4, Nkx2.5 and a-MHC at mRNA level in BMMSCs were detected by Real-time quantitative PCR at 1, 2, 4 weeks after routine culture. The consequences showed that,cells in each group did not express a-MHC, compared with the blank group, FGF-2 and Wnt-11 induced group cells could express GATA-4, Nkx2.5 in the first week, and then the expression of them was decreased in the second week, then increased in the fourth week; the expression of GATA-4, Nkx2.5 in the combination of FGF-2 and Wnt-11 induced group was slight, and the followed 2w and 4w was gradually increased, in the fourth week, gene expression in the combination induced group was significantly higher than that induced by each individual induction group(P<0.05).Via this experiment, It could draw a conclusion as follows:1.BMMSCs could differentiate into cardiomyocyte-like cells by the induction of FGF-2, Wnt-11 and the combination of them in vitro.2.Compared with the inducing effects of FGF-2 and Wnt-11, the combination of them was more effective in the procedure of differentiation of BMMSCs into cardiomyocyte-like cells. |
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