Research SDF-1 / CXCR4 Regulation Tip Endothelial Cells Into Vascular Effects Of The Molecular Mechanism

Part I Isolation 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 after4days culture and were able to form cells colonies after7days. The positive rates of CD34,CD133and VEGFR of ex vivo expanded endothelial progenitor cells were (66.3±2.14)%、(79.8±1.47)%.(94.6±1.93)%. After21days, the pebble-like cells spread the filopodia and connect 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. The effects of SDF-1and AMD3100on migration of Tip vascular endothelial cell[Abjective] To study the influence of SDF-1/CXCR4biological axis to the biological behavior of Tip vascular endothelial cells, this experiment aimed at through the application of certain concentration of chemokines SDF-1and AMD3100, as the inhibitor of CXCR4receptor, and study the Tip vascular endothelial cells in vitro so as to observe the effects of SDF-1and AMD3100on migration of Tip vascular endothelial cell. This could provied more evidences for treating Lower limb ischemia by Tip vascular endothelial cells.[Methods] The cultured peripheral blood endothelial progenitor cell in vitro were isolated and purified from human peripheral blood, and the cells were differentiated into vascular endothelial cell. After21days, the cells were cultivated with different concentrations of SDF-1(20ng/ml,50ng/ml,100ng/ml) and AMD3100for24hours. The abilities of migration of Tip vascular endothelial cells derived from peripheral blood endothelial progenitor cell were measured by the methods of transwell migration.[Results] The different concentrations group (50ng/ml>100ng/ml) of SDF-1can improve the migration of Tip vascular endothelial Cells, the OD were higher than the control group(P <0.05). the migration of the Tip endothelial Cells were not significant between the group of the concentration of20ng/ml with SDF-1and the control one(P>0.05); The migration of Tip vascular endothelial Cells were reduced by AMD3100alone、50ng/ml SDF-1was inhibited completely with10ng/ml AMD3100and100ng/ml SDF-1was inhibited completely with10ng/ml AMD3100,However, the OD of50ng/ml SDF-1was inhibited completely with10ng/ml AMD3100is lower than that of100ng/ml SDF-1(P<0.05).[Conclusion] The concentration of50ng/ml and100ng/ml with SDF-1can improve the migration of Tip vascular endothelial Cells, but this efects were blocked by the antagonist AMD3100.The migration of the Tip endothelial Cells obviously decrease with the CXCR4inhibitor AMD3100. The SDF-1/CXCR4signal pathway regulate the migration of the Tip endothelial Cells.