Study On The Effect Of Netrin-1Gene On Peripheral Blood Endothelial Progenitor Cells Derived Vascular Endothelial Cells And Its Mechanism

Objective:To discussion the separation, culture, identification of human peripheral blood endothelial progenitor cell and differentiation into mature endothelial cells, researching the conditions and biological activity of inducing culture in vitro, to provide further experimental research required cell source. Observation the endothelial progenitor cells differentiate into endothelial cells and the process of TIP formation of vessel structures through the factor of Netrin-1.Methods:Human peripheral blood mononuclear cells are isolated by the way of Ficoll density gradient centrifugation, which are inoculatede in the vaccination in package that is someone fiber connection protein cultivation in the bottle. Observed the morphology of cells under a microscope,and every3-4days for a liquid,the adherent cells were collected after seven days of culture, flow cytometry instrument testing surface markers to identify cells mainly for endothelial progenitor cells after induction. To detection the expression of mature endothelial cells’ vWF,eNOS gene by RT-PCR after4weeks. Cells collect adherent endothelial,were randomly divided into5groups:in the medium, to join the final concentration of0ug/ml,10ug/ml,50ug/ml,100ug/ml,500ug/ml netrin-1train for24hours. To observe Netrln-1cell proliferation ability; Under the action of Netrin-1lumen formation ability; Different concentrations netrin-1effects on endothelial cell proliferation.Results:(1) people of endothelial progenitor cells from peripheral blood in vitro culture and amplification:isolated peripheral blood mononuclear cells in culture after48h stick wall, in the fourth day of visible spindle cells and cell clusters, clusters of cells form including in the middle of the circle, spindle cells surrounding the cells and its structure is similar to blood island, spindle cells both individual cells and cable structure.7days in culture.(2)the cells cultured in vitro with the characteristics of the endothelial cells:to observe whether adherent cells with endothelial cell phenotype. We conducted vwF and FLK1, CD31, CD34immunohistochemical detection of endothelial cell surface markers, the results showed vwF, FLK1and CD31positive staining, positive rate>95%. The reports and Murohara similar, CD34in7days after showing negative or weakly positive results, to cultivate positive staining of10-14days. Culture cell fluorescent chemical test results show that>80%of cells show positive staining, prove that these cells can swallow ac-MIDI and combined with UEA-1, consistent with endothelial cell function.(3) There are the character of progenitor cells in vitro cultured cells, attached with a cell in the7d after the PE-AC133immunofluorescence test (MACS) tag, cytoplasm results showed>80%of cells show red fluorescence, positive staining.(4)the role of Netrin-1in cell proliferation abilityWhen the concentration of cultivating Netrin-1in less than500ng/ml, promote EPC proliferation. The Netrin-1has the strongest when the concentration of50ng/ml; And when the Netrin-1in100-500ng/ml, the inhibition of EPC proliferation (P<0.05).(5)the EPCs migration ability under Netrin-1factorObserved under light microscopy cellulose membrane on the surface of the cell, the nucleus is blue, the concentration of less than50ng/ml. The Netrin-1that the migration ability of EPCs, and in a certain concentration range of EPC migration ability is concentration dependent, strongest in1000ng/ml, and when the concentration in100-500ng/ml. Instead of EPC migration ability were inhibited (P <0.05).(6) The formation ability of EPCs by influens Netrin-1factorNormally, EPCs in covered by Malrigel cultivation in the plate after18h, a large number of dendritic structures in microscopically many sample into tubular shape concentration below50ng/ml, the netrin-1in the formation of the dendritic structure has no effect (P>0.05), and when the concentration of100-500ng/ml, the dendritic structure is significantly reduced, not seen tubule formation (P<0.05).Conclusion:Potential of proliferation and differentiation of endothelial progenitor cells can be successfully isolated from human peripheral blood, which can be expanded and induced into mature endothelial cells through specific induced in vitro cultivation system. Netrin-1not only can promote the cell culture derived endothelial progenitor peripheral blood vascular endothelial cells in vitro.but also can inhibit angiogenesis, mainly depends on the concentration of the injected dose.