Effects Of NGF On Endothelial Progenitor Cells Of Cerebral Infarction Patients And Its Mechanisms

ObjectiveTo master expertly in isolation of mononuclear cells(MNCs) from Human peripheral blood by Ficoll density gradient centrifugation and to induct and different endothelial progenitor cells (EPCs) . Find a way to gain endothelial progenitor cells and to establish a consistence foundation to the next study.Methods(1) The sterility anticoagulation under-pressure tube with EDTA was used to draw blood 10ml from ulnar vein of healthy adult and MNCs were isolated from Human peripheral blood by Ficoll density gradient centrifugation.(2) MNCs were cultured on the six well fibronectin-coated cell dishes and induced to EPCs. The change of the cellular shape was recorded after isolation at( 4days,7days,14days).(3) EPCs were characterized as Dil-acLDL/FITC-UEA-1 double positive cell and detected by laser scanning Confocal microscope (LSCM).EPCs were further documented by demonstrating the expression of KDR,CD133 and CD34 with flow cytometry.(4) EPCs were cultivated on the ECMatrixTM . After 21 days, the shape of the vessels was observed.Results(1) The character of EPCs appearance: cells that just isolating from blood can't adherence and the shape of those cell appears round. At 4days, the round cell transform into ellipse or short Fusiform. At 7days, lots of cell colony were observed , round cell at the middle of cell colony and Fusiform cell grow outside from center to surrounding. At 14days, cells connected with each other as trabs.(2) The isolating cells were stained as Dil-acLDL/FITC-UEA-1 double positive cell detected by LSCM.(3) EPCs were further documented by demonstrating the expression of KDR,CD133 and CD34 with flow cytometry. CD34 to occupy (32.15±8.68)%, CD133 occupy (18.73±7.12)%, KDR occupy (69.45±8.21)%. Further, the cells isolating from human peripheral blood were certified as EPCs.(4) After 21 days, the shape of the vessels on the ECMatrixTM glue was observed.Conclusions:Ficoll density gradient centrifugation is a available method to isolate and identify endothelial progenitor cells from Human peripheral blood. It is a reliability way to gain endothelial progenitor cells and to establish a consistence foundation to the next study. ObjectiveTo investigate the number and adhesion,migration, in vitrovasculo genesis capacity of endothelial progenitor cells (EPCs) from cerebral infarction patients.Morever,the effect of nerve growth factor on the number and funcation of EPCs were evaluated.Methods(1) To evaluate the number and adhesion,migration, in vitrovasculo genesis capacity of endothelial progenitor cells (EPCs) from cerebral infarction patients(2) the effect of nerve growth factor on EPCs from peripheral blood :EPCs were incubated with nerve growth factor in a series of concentrations (20ug/L,40ug/L,60ug/L,80ug/L) for 24 hours to choose the one affect the number and function of peripheral EPCs. EPCs were incubated with 60ug/L nerve growth factor for various time(0h,6h,12h,24h,48h) .(3) the effect of nerve growth factor on EPCs from peripheral cerebral infarction patients:EPCs were incubated with nerve growth factor 60ug/L for 24 hours, then evaluate the number and function of EPCs.EPCs proliferation were assayed with MTT. EPCs adhesion assay was performed by replating MNCs on culture dishes, and then the effect cells were counted. Migration were assayed with Boyden chamber. And in vitro vasculogenesis activity was assayed in vitro vasculogenesis kit.Results(1) The number and functional activities of EPCs such as migration,adhesion,proliferation and in vitro vasculogenesis activity were significantly impaired in cerebral infarction patients.(2) Treating the EPCs with nerve growth factor, the number and functional activities of EPCs such as migration,adhesion,proliferation and in vitro vasculogenesis activity were significantly improved. 60ug/L nerve growth factor got to the peak after 24h (p < 0.01).(3) Pretreated EPCs of cerebral infarction patients with 60ug/L nerve growth factor, the number and migration,adhesion,proliferation and in vitro vasculogenesis activity of EPCs were increased significantly.ConclusionNerve growth factor increase the number of cultured EPCs and improve EPCs biological characteristics of cerebral infarction patients. ObjectiveTo investigate the effect of NGF on the expression AKT and eNOS, in cultured endothelial progenitor cells (EPCs) from peripheral blood of cerebral infarction patients, in order to study the mechanism of NGF on EPCs number and activity.Methods(1) EPCs of cerebral infarction patients were incubated with or without NGF LY294002 or L-NAME for 24 hours Expression eNOS, phosphorylation and eNOS were measured by Western Blot.(2) EPCs of cerebral infarction patients were incubated with NGF LY294002 or L-NAME for 24 hours Expression PKB eNOS,phosphorylation of PKB and eNOS were measured by Western Blot.(3) EPCs of cerebral infarction patients were incubated with NGF +LY294002 or L-NAME for 16 hours to To evaluate the number , function and NO of peripheral blood EPCs of cerebral infarction patients. EPCs proliferation were assayed with MTT. EPCs adhesion assay was performed by replating MNCs on culture dishes, and then the effect cells were counted. Migration were assayed with Boyden chamber. And in vitro vasculogenesis activity was assayed in vitro vasculogenesis kit. Concentration of NO were measured using Griess Reaction in cell culture supernatants.(4) EPCs of cerebral infarction patients were incubated with or without NGF,Concentration of NO were measured using Griess Reaction in cell culture supernatants.Results(1) The expression of eNOS phosphorylation decreased in EPCs from peripheral cerebral infarction, NGF inproved eNOS phosphorylation without influence total eNOS. L-NAME inhibited phosphorylation of PKB, and also abolished the effect of NGF on L-NAME phos phorylation.(2) The expression of PKB phosphorylation decreased in EPCs from peripheral cerebral infarction, NGF inproved PKB phosphorylation without influence total PKB. LY294002 inhibited phosphorylation of PKB, and also abolished the effect of NGF on PKB phosphorylation(3) The number and functional activities of EPCs such as migration,adhesion,proliferation and in vitro vasculogenesis activity were significantly decreaseded in cerebral infarction patients. Treating the EPCs with either LY294002 or L-NAME , the number and functional activities of EPCs were significantly impaired comparable with these untreated. At same time the generation of NO decreased.(4) Concentration of NO in EPCs culture supernatants decreased signicantly in cerebral infarction patientsConclusionDownregulation of PKB/eNOS pathway and decrease in NO generation was observed in EPCs in cerebral infarction patients,which appears to mediate the impairment number and functional activities of EPCs such as migration,adhesion,proliferation and in vitro vasculogenesis activity. NGF upreguation of PKB/eNOS phosphorylation and NO generation may potentially contributed to improvement of number and capacity of EPCs in in cerebral infarction patients.