Molecular Mechanism Of MicroRNA - 221 Regulating The Vascular Effect Of Tip Vascular Endothelial Cells

Part IIsolation and differentiation of human endothelial progenitor cells, vascular endothelial cells and Tip vascular endothelial cells[Abjective] To investigate the methods of isolation and cultivation of human endothelial progenitor cells (EPCs). To Research the culture conditions of human endothelial progenitor cells in vitro and biological characteristics and confirm the peripheral blood is the another idear source of human endothelial progenitor cells except the bone marrow. The human endothelial progenitor cells were differentiated the vascular endothelial cells and Tip vascular endothelial cells. This can provide the cells for next experiment and a reliable basis for angiogeneisis.[Methods] Peripheral blood mononuclear cells were isolated by density-gradient centrifugation and cultured in endothelial growth medium-2in the presence of vascular endothelial growth factor. Observe the morphology of the cells every day by microscopy. The cells were detached and indentified by endothelial-specific markers at day7. The expression of CD34,CD133and VEGFR were determinded by flow cytometry. After21days, the vWF and eNOS were determinded by reverse transcriptase-polymerase chain reaction(RT-PCR).[Results] Most of the cells attached after4-day culture and were able to form cell colonies after7days. The positive rates of CD34,CD133and VEGFR of ex vivo expanded endothelial progenitor cells were (68.2±1.15)%、(72.3±3.42)%、(95.6±2.01)%. After21days, the pebble-like cells spread the filopodia and connected each other. RT-PCR demonstrated the presence of vWF and eNOS mRNA.[Conclusion] Endothelial progenitor cells could be successfully isolated and cultured from perpheral blood mononuclear cells. The human endothelial progenitor cells also were differentiated the vascular endothelial cells and Tip vascular endothelial cells. Part IIThe second part of peripheral blood Tip vascular endothelial cell migration and proliferation[Abjective] To study the biological effects on vascular endothelial cells Tip microRNA-221biological axis, this experiment aimed at small nucleotide application of a certain concentration of-221, Tip, vascular endothelial cells cultured in vitro as research object, observe the tiny nucleotide-221(microRNA-221) Tip in peripheral blood endothelial cell migration and proliferation, to lay the foundation for clinical practice Tip endothelial cell transplantation for the treatment of angiogenesis.[Methods] after21days of culture of adherent endothelial cell Tip collected in the first part of the experiment, the cell suspensions were divided into5groups:control group. A:B:10ug/L miR-221, C:20ug/L miR-221, D:50ug/L miR-221, E:100ug/L miR-221+10mg LCXCR4antibody. The first use of Lipofectamine2000(Invitrogen) was transfected into miRNA precursor;The effects of Tip vascular endothelial cell migration and proliferation, after using Transwel1、MTT to test different concentrations of miR-221on peripheral blood derived endothelial progenitor cells.[Results]the B:lOug/L miR-221, C:20ug/L miR-221, D:50ug/L miR-221, E:100ug/L miR-221+10mg LCXCR4antibody, migration and proliferation in four group Tip were induced to vascular endothelial cells, migration and proliferation ability was significantly higher than the control group (P<0.05), and the transfection of miR-221into the cell concentration, influence of Tip endothelial cell migration and proliferation ability of the cells to endothelial progenitor peripheral blood is bigger.[Conclusion]10ug/L miR-221,20ug/L miR-221,50ug/L miR-221,100ug/L miR-221+10mg LCXCR4antibody in the four group could promote the ability to migrate and proliferate endothelium of Tip in peripheral blood in vitro; so that miR-221may be involved in the regulation of Tip in vitro migration and proliferation of vascular endothelial cells.