Isolation, Culture And Induced Differentiation On Rat Bone Marrow Mesenchymal Stem Cells And Immune Regulatory Effect On T Lymphocyte

BACKGROUND: Besides hematopoietic stem cells in bone marrow, there are another kind of stem cells, which arise from the mesenchymal tissue, so called mesenchymal stem cell(MSC), also known as bone marrow stromal stem cells. Recently more and more studies provide evidence that bone marrow mesenchymal 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. In addition can be harvested easily from bone marrow with standardized isolation techniques and have a high proliferative potential in culture , and when autologouse transplanted, there exist no immunorejection. So recently more and more attention has been paid to the treatment for many diseases using bone marrow mesenchymal stem cells. This will open the new way for rare animal's treatment and the extension.In this process, an important first step required for tissular or organic regeneration and reparation by circulating stem cells involves adhesion of these cells to vascular endothelial cell(VEC) or extracellular matrix(ECM). It can be concluded that cell adhesion molecule plays an important role in intercellular interaction. So it is extraordinary important to investigate the biological and molecular basis for an intercellular interaction between MSCs and VECs or ECM. In recent years, more and more research also shows that the MSCs is immune regulation of their immune suppression in the area which can be expected with the theoretical significance and value. So we design and do the experiments including the following two parts:PART 1: THE ISOLATION, CULTURE AND EXAMINATION OF THE SURFACE PHENOTYPE ON RAT BONE MARROW MESENCHYMAL STEM CELL IN VITROObjective:To investigate the more effective methods of isolation, culture of mesenchymal stem cells (MSCs) , and examine the surface phenotype of MSCs.Methods:MSCs were separated from rats bone marrow by plastic adherence methods and purified by controlling the time of digestion combied with adhesion separation , and proliferated in culture flasks that were coated withⅠcollogen. The morphology of MSCs were studyed with phase contrast microscope; cell cycles of the forth generation of MSCs were tested by flow cytometry and the phenotype of MSCs were identified by using immunocytochemical methods .Results: Primary cultured MSCs were oval,spindle-shaped or polygonal,and adhered to plastic surface within 24 h and reached 90% confluence within 7～8 days.After purification and proliferation, they were uniformly long spindle-shaped form and passaged every five days. The adhesion rate within 24 hours was all. The flow cytometry showed 80% cells of the forth generation of MSCs were at G0/G1 phase. Immunocytochemistry showed MSCs were positive for CD29,CD105,CD166,VLA-4 and P-selectin,while negative for CD34 and CD45.Conclusion: The MSCs obtained from our experiments were more purified, expanded more rapidly and expressed some cell adhesion molecules. The protocol should make it possible to undertake a large number of experiments with MSCs in future application. PART 2: IMMUNE REGULATORY EFFECT OF RAT BONE MARROW MESENCHYMAL STEM CELLS ON T LYMPH- OCYTEObjective:To investigate the immune regulatory effects of Rat bone marrow mesenchymal stem cells on alloantigen T lymphocyte in vitro.Methods: MSCs were separated from rats bone marrow by plastic adherence methods and purified by controlling the time of digestion combied with adhesion separation , and proliferated in culture flasks that were coated withⅠc ollogen.With inverted phase contrast microscope observe MSCs shap, with Flow cytometry detect MSCs molecular marker. T lymphocytes were obtained by using nyloneolumn,with flow cytometry detect CD3 molecular expression.T lymphoeytes proliferation was measured by MTT method.Result: MSCs were homogenous population,The cell curve showed MSCs had a good ability of proliferation,MSCs were positive for CD44,CD13 Mononuclear cells which purified with nylon column high express CD3 molecule T lymphoeytes proliferation was suppressed by MSCs or supernatant signifieantlyConclusion: MSCs and T lymphoeytes obtained in this study were reliable MSCs or supernatant suppress T lymphocyte to proliferation.