The Expression And Significance Of Cell Adhesion Molecules On Rat Marrow Mesenchymal Stem Cells

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 cells(MSCs), 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.Now, the two methods to treat for many diseases using MSCs:1) autoallergic mobilization, 2) cell transplantation. 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). So it is extraordinary important to investigate the biological and molecular basis for an intercellular interaction between MSCs and VEC or ECM. It can be concluded that cell adhesion molecule plays an important role in intercellular interaction. However, the expression and significance of cell adhesion molecules on bone marrow mesenchymal stem cells is still not readable. So we design and do the experiments including the following two parts:PART 1: BIOLOGICAL EFFECTS OF THE EXTRA CELLULAR MATRIX ON RAT BONE MARROW MESENCHYMAL STEM CELLs IN VITROObjective: To observe the biological effects of polylysine on rat marrow mesenchymalstem cells(rMSCs), and investigate the optimal condition of isolation, culture andexpanding of rMSCs.Methods: MSCs were isolated from rats bone marrow by plastic adherence methodsand purified by controlling the time of digestion combied with adhesion separation.The cells were respectively plated in culture flasks that were coated with polylysine ornot. Then we studied the morphology of the cells with phase contrast microscope,measured the number of adhesive cell, drawed the growth curve and examined thecomprising cell cycle by a fluorescenceactivated cell sorter and the phenotype ofMSCs were identified by using immunocytochemical methods .Results: The adhesion and proliferation of MSCs growing on the polylysine weremore rapid than the controlled group, the morphous of cells were more typical and thegrowing state of cells were more vigorous. And after purification and proliferation, thecells were uniformly long spindle-shaped form.Conclusion: (1) This polylysine of extracellular matrix can significantly increase theadhesion and proliferation capacity of ex vivo expanded MSCs, which indicates theamplification and cultivation of MSCs in vitro became more simply and efficiently. (2)The MSCs obtained from our experiments were more purified ,and expanded morerapidly , which make it possible to undertake a large number of experiments withMSCs in future application .PART 2: THE EXPRESSION AND SIGNIFICANCE OF VCAM-1 and ICAM-1 ON RAT BONE MARROW MESENCHYMAL STEM CELLS Objective: To investigate the expression of VCAM-1 and ICAM-1 in rMSCs , andstudy the effect of TNF-α on the expressional level of cell adhesion moleculesVCAM-1 and ICAM-1 in rMSCs; in orde to investigate the mechanisms of thetransplantion, migration and adhesion of MSCs.Methods: MSCs were separated from rats bone marrow by plastic adherence methods .Then rMSCs were divided into two groups: the cells of group one were respectivelytreated with TNF-a(10 ng/ml) forl2 h ;the other group was regarded as control. TheVCAM-1 and ICAM-1 protein were identified by using immunocytochemical methods ,the rate of antigen were tested by flow cytometry and their mRNA expression levelswere measured with RT-PCR.Results: Immunocytochemistry, flow cytometric analysis and RT-PCR all showed thatrMSCs expressed low levels of VCAM-1 while high levels of ICAM-1, and theexpression levels of VCAM-1 were up-reguated remarkably by TNF-α. Whencompared with VCAM-1 expression, the levels of ICAM-1 were not affected byTNF-α.Conclusion: 1.The rMSCs expressed levels of VCAM-1 were very low whereas cellsexpressed high levels of ICAM-1. 2.The rMSCs express more VCAM-1 afterstimulation with these cytokines (TNF-α), and these cytokines have almost no effecton ICAM-1 expression levels.