The Multiplication And Differentiation Into Endothelial Cells Of Mesenchymal Stem Cells In GFP-expressing Mouse In Vitro

Seed cell is one most important problem on vascular tissue engineering studying. Being a type of seed cell ,endothelial cell acts an significant character in vascular tissue engineering in the present study.How to obtain sufficiency endothelial cells is a recently subject. The multilineage potential of Mesenchymal stem cells (MSCs), their ability to differentiate into a wide variety of lineages and to elude detection by the host's immune system, and their relative ease of expansion in culture make MSCs a very promising source of stem cells be a new source of seed cell of vascular tissue engineering. Differentiated mechanisms and induced condition of MSCs is not really clear. In multistandpoint, the differentiation of mesenchymal stem cells have a intimate relation with microenvironment. It's maybe various factors of microenvironment which stimulate MSCs differentiation. Current study reveal that vascular endothelial growth factor and shear stress can accelerate differentiation from MSCs to endothelial cells. However,it is still a question that which method we will adopt to induce reasonably and operatively.Objective This study aims to explore the isolation,multiplication and differentiation into endothelial cells of green fluorescent protein(GFP)-expressing mouse bone marrow MSCs in vitro.Methods (1) Studying with GFP mouse,GFP mouse bone marrow was aspirate from thighbone by aseptic technique.MSCs were then obtained and cultured in DMEM/F12 in vitro. Then, MSCs were amplificated and transferred of culture. (2) MSCs were induced by vascular endothelial growth factor (VEGF) directly and transfected plasmid contained VEGF cDNA to differentiate into endothelial cells, and were detected by immumofluorescence method, and Calculated the proportion of male cell. Compared the results of two methods,and chosen the effective method.(3) The experiment were divided into three groups: (1)First group cells were exposed to shear stress for 24 h .(2)Secondgroup cells were induced by transfected plasmid contained VEGF cDNA. ?Third group cells were induced by transfected plasmid contained VEGF cDNA,and then exposed to shear stress for 24 h.All three groups's cells were cultured for 1,3,5,7,10 days after they were induced,then, detected by immumofluorescence method, and Calculated the proportion of male cell. Compared the results, and chosen the effective method.Results (1) The number of MSCs increased four to eight-fold with each multiplication passage. 7.0 X 103 primary MSCs multiplied in three passage to reach a number of 1.4 X 108.Cobble-stone-like structure was observed at 70-80% confluence of cells.The result demonstrat that MSCs of the GFP mouse bone marrow can multiply vigorously. (2)When MSCs were induced by VEGF directly, proportion of male cell of vWF were rising step by step as time goes by.The transformation efficiency arrive to a tip while MSCs being induce for three days. Detected by immumofluorescence method,we found that proportion of male cell of vWF were 2.1%,23.3%, 25.7%,23.5% and 21.8%,when MSCs were induced for 1,3,5,7,10 days. When MSCs were transfected plasmid contained VEGF cDNA to differentiate into endothelial cells, proportion of male cell of vWF were rising step by step as time goes by. By immumofluorescence method, we found that proportion of male cell of vWF were 1.3%,5.6%,14.4%,23.5% and 33.1%,when MSCs were induced for 1,3,5,7,10 days.Comparing with the transformation efficiency of two methods, we found that the transformation efficiency of the method that transfected plasmid contained VEGF cDNA were higher than the method that induced by VEGF directly later.All of the results above demonstrat that MSCs of the GFP mouse bone marrow can be induced to differentiate into endothelial cells by VEGF in vitro.Effect of conversion would decreased because of decreasing of VEGF's concentration.However,the method that transfected plasmid can make cells express VEGF ,and it can stimulate MSCs differentiating into endothelial cells utility in long-term. (3) Detected by immumofluorescence method,we found that MSCs induced by shear stress only can express male cell of vWF cultured 3days after.It's prove that MSCs would differentiated into endothelial cells induced by shear stress. Cells that induced by transfected plasmid contained VEGF cDNA and shear stress were observed after cultured 1,3,5,7,10 days.By immumofluorescence method, it show that proportion of male cell of vWF were 2.1%,7.3%,20.8%,38.0% and 63.2%. Comparing with control, discrepancy is marked (P<0.01)o The result demonstrat that shear stress could effectivelypromote MSCs differentiating into endothelial cells.Conclusion (1) MSCs of the GFP mouse bone marrow can be induced to differentiate into endothelial cells by VEGF in vitro. Effect of conversion would decreased because of decreasing of VEGFs concentration.The transformation efficiency of the method that transfected plasmid contained VEGF cDNA were higher than the method that induced by VEGF directly.(2) Shear stress markedly increased MSCs differentiated into endothelial cells.