Antiglioma Activity Of Genetically Modified Endothelial Progenitor Cells Via Inhibiting Angiogenesisin Vitro And In Vivo

PART ONE:Culture and identification of endothelial progenitor cells from cord blood with CD133 immunomagnetic sortingObjective To investigate the methods of isolating and culturing endothelial progenitor cells(EPCs)from cord blood,which have the high potential of proliferation,tube formation and migration. Methods CD133~+ cells were selected from fresh cord blood mononuclear cells by magnetic activated cell sorting system(MACS),and cultured in EGM-2 MV culture medium.EPCs were studied by morphology,cell markers and functions.Results On day 7,the adherent cells exhibited the small clonal morphology;On day 21,the clonies expanded,confluenced, and displayed a typical "cobblestone" morphology.After 14 days of culture,more than 90%attached cells took up Dil-ac-LDL,and bound FITC-UEA-1(double positive fluorescence).Compared with the original, cell markers CD133 and CD34 decreased significantly,while CD31 increased significantly.EPCs were different from mature endothelial cells in terms of proliferation,tube formation and migration,which was proved by comparison between EPCs and human umbilical vein endothelial cells (HUVECs).Conclusion EPCs,with the capacity of proliferation,tube formation and migration,can be cultured from cord blood mononuclear cells using CD133~+ MACS.PART TWO:Mechanism of endothelial progenitor cells in the angiogenesis of glioma cellsObjective To investigate the mechanism of endothelial progenitor cells(EPCs)in the angiogenesis of glioma cells.Methods After EPCs were treated with the supematant from U87 glioma cells or VEGFR-2 siRNA,tube formation,migration,VEGFR-1,VEGFR-2,Akt and ERK expression,and MMP-9 activity and expression,were determined by tube formation assay on matrigel,migration assay, Western blot analysis,and gelatin zymography analysis,respectively. Results The supernatant from U87 glioma cells induced EPCs tube formation,and migration.The supernatant from U87 glioma cells triggered a marked increase in VEGFR-2 expression,with minimal effect on VEGFR-1 expression.Moreover,MMP-9 activity and expression were up-regulated.VEGFR-2 siRNA inhibited VEGFR-2 expression in EPCs, cell migration and tube formation on matrigel.MMP-9 activity and expression were decreased by VEGFR-2 siRNA.The Akt and ERK signal pathway phosphorylations were involved in EPCs angiogenesis mediated by VEGFR-2.Conclusion Glioma cells induced EPCs angiogenesis via up-regulating VEGFR-2 mediated by MMP-9,Akt and ERK signal pathways.Objective To explore the potential of HSV-TK tranduced endothelial progenitor cells(EPC-TK)as angiogenesis-targeted vector in the glioma treatment in vitro and in vivo.Methods Sensitivity of EPC-TK to GCV was measured for 48 h in the presence or the absence of increasing concentrations of GCV.EPC-TK were mixed with HUVECs,U87 or U251 cells at varying ratios for GCV,then survival cells were counted using the MTT assay,and apoptotic cell death was determined by annexin-V and phosphatidylserine(PI)staining.EPC-TK were injected into the nude mice model of glioma xenograft by tail vein, and the changes of tumor volume and tumor vasculature were observed. Results EPC-TK were sensitive to GCV in a concentration-dependent manner.GCV killed most EPC-TK and reduced the number of viable other cells in a cell:cell ratio-dependent and time-dependent manner. EPC-TK obviously inhibited tumor growth and angiogenesis.Conelusion EPC-TK can exert a potent antiglioma effect via inhibiting angiogenesis.