| Recently more and more attention has been paid to the study about stem cells. The defining characteristics of stem cells are self-renewal and the ability to differentiate into one or more specialized cell types. According to their capacity of differentiation, stem cells have been divided into three major groups: totipotential stem cells, multipotential stem cells and unipotential stem cells. It was believed that only embryonic stem cells derived from the inner cell mass could differentiate into any cell type, but unipotential stem cells, also called adult stem cells or tissue-specific stem cells, could only differentiate into cells of the tissue of origin. However, several studies now provide evidence that tissue-specific stem cells may be able to give rise to a variety of differentiated cell types found in embryonic germ layers. This founding is important for the developing of cytobiology theory and the treatment for many diseases.The two most attractive tissue-specific stem cells are: 1) hematopoietic stem cells, which must provide a continuous source of progenitors for red cells, platelets, monocytes, granulocytes, lymphocytes; and 2) bone marrow stromal stem cells (BMSCs), also known as mesenchymal stem cells or colony-forming-unit fibroblasts, which arise from the complex array of supporting structures in marrow. Recent workhas shown that BMSCs have the capacity to differentiate along a number of lineages under defined conditions in vitro and in vivo. Because BMSCs can be harvested easily from bone marrow with standardized isolation techniques and have a high proliferative potential in culture, also BMSCs are capable of differentiation to a wide variety of cell types, and when autologous transplanted, there exist no immunorejection. So BMSCs can be used to treat many dieases.In this dissertation, we studied the isolation, culture and induced differentiation of rat BMSCs, especially the proper condition for cardiomyocytes differentiation in vitro and in vivo. Also we established myocardial infarction model in rats, then autologous transplanted BMSCs to the model to study the existing, differentiation, and vasculogenesis of transplanted cells.1 Isolation, culture and expanding of BMSCs is the premise of the total experiment, so we carried out this part firstly. ( reference to chapter 1 of the dissertation)The number of BMSCs is estimated to be too few in bone marrow ( 2-5 BMSCs per 106 mononuclear cells), so it is important to expand and purify the BMSCs for the following experiments. BMSCs were isolated from adult rats using density centrifugation and anchoring culture, then cultured in low-glucose DMEM supplement with 10% fetal bovine serum (FBS) for expanding. Colonies of BMSCs were formed at 4th day at primary culture, at about 7th day, BMSCs got together and the number of division cells decreased, then were subcultured by 0.25% trypsine-0.02%EDTA. BMSCs were purified at 3rd generation. The passage BMSCs proliferated fast, and could be subcultured about every 5 days. In later generation, BMSCs grew slowly.The antigenic profile displayed by BMSCs is not unique, thus it cannot be associated with a particular cell phenotype. Instead, it suggests that BMSCs borrow features from mesenchymal, endothelial, and epithelial cells. So there exists no directly means to identify BMSCs. Now BMSCs are identified by their potential of differentiation into multiple cells.2, BMSCs have been focus in biotic science in recent years because of their ability to differentiate into multiple lineage cells. Differentiation is the process in whichimmature cells develop into specific cells with specific morphology and function. It is the result of different genes expression of cells containing identical genome. Because gene expression demands certain condition, cell differentiation is regulated by such elements as cell interaction and environmental factors, such as growth factors, hormones and chemical drugs. In this experiment, we emphasized the transdifferentiation potential of BMSCs.To investigate whether BMSCs can give rise to mesodermic cells, BMSCs were cultured either in osteogenic ( dexamethasone, 3 -glycerophosphate, ascorbic acid) or adipogenic (dexamethasone, insulin) medium. It was found that when BMSCs were incubated in the osteogenic medium, the Alkaline Phosphates (ALP) activity was enhanced and formed mineralized nodules. However, when BMSCs were grown in the adipogenic medium, adipocytes were detected and the ALP acativity declined. It showed that low concentration of dexamethasone can induce BMSCs differentiate into osteoblasts, while high concentration dexamethasone into adipocytes. ( reference to section 1, 2 of chapter 2 )To further explore the transdifferentiation potential of BMSCs, we focused on the neuronal inductive experiment. BMSCs were pretreated in DMEM/FBS/ P -mercaptoethanol (3 -ME) for 24 hours. Neuronal differentiation was induced with DMEM/ 3 -ME. Cells were fixed for immunocytochemistry at time ranging from 1 to 5 hours. Within 1 hours of exposure to serum-free inductive medium, changes in morphology of some of the BMSCs were apparent, above 70% BMSCs progressively assumed neuronal morphological characteristics over 5 hours. The changed cells expressed neuron-specific enolase, but not glial fibrillary acidic protein. This experiment showed that BMSCs could be induced differentiation exclusively into neurons, (reference to section 2 of chapter 2)H> Although terminally differentiated cardiomyocytes have shown some evidence of mitotic division in the adult heart, the rate of proliferation is minute and cannot meet the demand for tissue regeneration, and there exist no resident stem cells in the heart. Then loss of cardiomyocytes leads to regional contractile dysfunction, and necrotized cardiomyocytes in infarcted ventricular tissues are progressively replaced byfibroblasts form scar tissues. Then repairing and regeneration ischemic or damaged myocardium is a promising approach to treat cardiac disease. We investigated the potential of BMSCs to undergo myogenic differentiation to test their potential use as a therapeutic tool for the treatment of myocardial infarction.The mechanisms to induce BMSCs to undergo cardiomyogenic are not fully understood. It is now assumed that the chemical induce in vitro and microenviroment in vivo play a significant role on the differentiation of these cells. To explore the optimal condition under which BMSCs can be induced to differentiate into cardiomyocytes in vitro, BMSCs were treated with different concentration of 5-azacytidine (5-aza) at different time. The result showed that the inductive effect of 5-aza had strict restriction of inductive occasion and concentration, also had relationship with cell density and state. The morphology of passage 3 BMSCs changed significantly when treated with 10 u mol/L 5-aza, and treated again after passaged. The treated BMSCs changed into spindle-shape and formed myotube-like structure, and can spontaneously beating, also stained positive for the cardiomyocyte-specific marker, showed the consistency of structure and function. This study showed that during the differentiation of stem cells, the inductive process not only depended on inductive effect of revulsant but also depended on the responsibility of induced cells, and still depended on temporal and spatial coordination between the the two aspects, (reference to section 1 of chapter3)It has been shown that microenviroment played significant role on the differentiation of stem cells. It is assumed that the specific molecules, growth factors, cytokines and interactions among stem cells, host cells and extracellular matrix are necessary, and they are presented to the stem cells in a temporal and sequential manner in order to induce the expression of related developmental genes in sequence and driving their differentiation into different cells. On the basis this theory, we implanted BMSCs labelled with BrdU into normal myocardium to test whether BMSCs could undergo milieu-dependent differentiation and express cardiomyogenic phenotypes in vivo. Four weeks after implantation, transplanted cells were found to be incorporated into the host myocardium and expressed cardiac-specific structural proteins. Wesuggested that the normal myocardial microenvironment may induce BMSCs differentiation into cardiomyocytes. ( reference to section 2 of chapter 3)Form above, we demonstrate that BMSCs are myogenically differentiated not only with 5-aza in vitro but also by normal myocardial microenvironment. BMSCs should be considered as an alternative transplante cell source to repair damaged myocardium.0 ^ BMSCs are of great therapeutic potential because of their ability to differentiate into multiple cell types. On the basis of induced differentiation of BMSCs into cardiomyocytes in vitro and in vivo, we autologous transplanted BMSCs into infarction myocardium to evaluated cardiomyogenic differentiation of BMSCs, their survival in myocardial scar tissue and peripherial normal myocardium tissue.Establishment a steady and repeatable myocardial infarction model is premise of this study, we used Wistar rats to perform thoracotomy and ligated the left decending coronary artery. 4 weeks later, the infarcted heart was larger than normal heart, the ischemic part was pale and ventricular wall was sunken, and necrotized cardiomyocytes in infarcted ventricular tissues were replaced by fibroblasts form scar tissues. It showed that we had successfully generated myocardial infarction model. ( reference to the section 1 of chapter 4)To further investigate whether implantation of autologous BMSCs resulted in sustained engraftment, myogenic differentiation, and vasculogenesis in rat myocardial infarction model, we established myocardial infarction model and harvested BMSCs from the same rat. After treated by 5-azacytidine and labelled with BrdU, treated and non-treated BMSCs were autologous transplanted into the center and the periphery of the infarct region. 4 weeks later, the heart was extracted. The existing and differentiation of the transplanted cells were identified by immunohistochemistry. The transplanted cells mainly located in the periphery of the infarct region and differentiaed into cardiomyocyte-like cells and vascular endothelial cells. While in the infarct region, only few transplanted cells existed and showed fibroblast-like morphology. The result of induced and non-induced BMSCs transplantation had no significantly difference. This study showed that BMSCs transplantation can be used to treat myocardial infarction for cellular cardiomyoplasty. But there needs an effective inductive project...
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