The Influence Of VEGF On Endothelial Components In Human Bone Marrow And Umbilical Cord Mesenchymal Stem Cells

Objective:1. To investigate the proportion of endothelial progenitor cell (EPC) and endothelial cell (EC) in human bone marrow mesenchymal stem cell (BM-MSC) and human umbilical cord mesenchymal stem cell (UC-MSC);2. To research the influence of Vascular Endothelial Growth Factor (VEGF) on the proportion of endothelial progenitor cell (EPC) and endothelial cell (EC) in MSC.Methods:1. Isolation and culture of UC-MSC:The fresh umbilical cord were minced into1-2mm fragments under sterile conditions, then were put into the bottom of Petri dish. The cells were cultured at37℃in a5%CO2atmosphere. After about a week, fibroblast-like cells reached about80%confluence. For passaging, confluent UC-MSC were detached with0.25%trypsin and reseeded at a density of5×104/cm2under the same culture conditions.2. Isolation and culture of BM-MSC:Bone marrow cells were separated over a Ficoll density gradient, and the mononuclear cells were harvested and washed with phosphate-buffered saline (PBS). Cells were cultured at37℃in a5%CO2atmosphere. After about two weeks, fibroblast-like cells reached about80%confluence. For passaging, confluent UC-MSC were detached with0.25%trypsin and reseeded at a density of5×104/cm2under the same culture conditions. 3. Experimental groups:the concentration of VEGF of the control group was Ong/ml, while the experimental group was10ng/ml.4. The proportion of CD34+CD133+and vWF+CD31+double positive cells in MSC of the fifth passage cultured cells were detected by flow cytometry.5. Wright’s staining was employed to observe the influence of VEGF on morphology of MSC.6. The expressions of CD34, CD133, CD31and vWF were detected by immuno fluorescence.7. qRT-PCR was carried out to detect the influence of VEGF on EC marker genes’ expression of MSC.Results:1. Some fibroblast-like cells were observed as early as one week after transferring fragments into plate, and they reached80%confluence with uniform fusiform appearance within15days. After2～3times’passaging the cells formed a monolayer of homogeneous spindle-like cells with a whirlpool like array.2. The results of the flow cytometric analysis showed that there were a small amount of EC and endothelial progenitor cells (EPC) in obtained BM-MSC and UC-MSC, after exposed to VEGF, the proportion of EC increased, while EPC decreased.3. Wright’s staining showed that the aspect ratio of MSC increased after exposed to VEGF.4. The Expression of EC related marker genes such as Tie-2and ecNOS upgraded, especially in UC-MSC.Conclusions:1. There were a small quantity of ECs and EPCs in cultured BM-MSC and UC-MSC;2. VEGF could increase the proportion and properties of EC in MSC.