Background Cellular repressor of ElA-stimulated genes(CREG) is homeostatic modulated gene,which regulate a number of cellular processes,including cell differentiation, motility and survival.Previous studies have demon...Background Cellular repressor of ElA-stimulated genes(CREG) is homeostatic modulated gene,which regulate a number of cellular processes,including cell differentiation, motility and survival.Previous studies have demonstrated that CREG was expressed in all three germ layers,suggesting that it might act as a vital regulator during embryonic developing.The aim of the present study was to investigate the role of CREG in an embryonic stem cell(ESC) differentiation model that recapitulates the developmental steps of vasculogenesis.Methods The ES cells were stably transfected either pCXN2-FLAG-CREG-IRES-EGFP plasmid or pDS1- shRNA-CREG plasmid to produce the CREG+/ES cells and CREG-siRNA/ES cells,respectively.Vasculogenesis was detected by whole mount immunostainings for CD31.Dil labeled acLDL staining assay was used to detect branching pseudopods in cultures in Matrigel.Real-time PCR and Western blot analysis were employed to determine expressions of VEGF and Flk-1.Results CREG +/ES-derived embryoid bodies(EBs) were found to form spontaneously a primitive vascular network after 6 days of differentiation.In contrast, wildtype EBs exhibit theirs vasculogenesis until 13 days of differentiation by whole mount immunostainings for CD31. CREG +/EBs developed more rapidly branching pseudopods at 9 days compared with that of wildtype EBs by Dil labeled acLDL staining assay.In contrast,CREG-siRNA/ES exhibits an undifferentiated morphogenesis associated with an increase in apoptotic cells in spite of being derived from LIF and feeder layers.Administration of CREG-siRNA/ES cells with recombinant CREG protein rescued the phenomena that CREG boosted vasculogenesis in a dose-dependent fasion. Mechanically,Real-time PCR and Western blot analysis revealed the expressions both VEGF and Flk-1 significantly in- creased in CREG+/EBs.Moreover,after treatment of CREG+ /EBs with neurtralizing antibody against VEGF,the rapid vasculogenesis was significantly repressed.Conclusions Our data strongely demonstrate that CREG play a pivotal role in accelerating vasculogenesis in development of ES cells. VEGF,as its important downstream effector,mediated this bio-function.展开更多
文摘Background Cellular repressor of ElA-stimulated genes(CREG) is homeostatic modulated gene,which regulate a number of cellular processes,including cell differentiation, motility and survival.Previous studies have demonstrated that CREG was expressed in all three germ layers,suggesting that it might act as a vital regulator during embryonic developing.The aim of the present study was to investigate the role of CREG in an embryonic stem cell(ESC) differentiation model that recapitulates the developmental steps of vasculogenesis.Methods The ES cells were stably transfected either pCXN2-FLAG-CREG-IRES-EGFP plasmid or pDS1- shRNA-CREG plasmid to produce the CREG+/ES cells and CREG-siRNA/ES cells,respectively.Vasculogenesis was detected by whole mount immunostainings for CD31.Dil labeled acLDL staining assay was used to detect branching pseudopods in cultures in Matrigel.Real-time PCR and Western blot analysis were employed to determine expressions of VEGF and Flk-1.Results CREG +/ES-derived embryoid bodies(EBs) were found to form spontaneously a primitive vascular network after 6 days of differentiation.In contrast, wildtype EBs exhibit theirs vasculogenesis until 13 days of differentiation by whole mount immunostainings for CD31. CREG +/EBs developed more rapidly branching pseudopods at 9 days compared with that of wildtype EBs by Dil labeled acLDL staining assay.In contrast,CREG-siRNA/ES exhibits an undifferentiated morphogenesis associated with an increase in apoptotic cells in spite of being derived from LIF and feeder layers.Administration of CREG-siRNA/ES cells with recombinant CREG protein rescued the phenomena that CREG boosted vasculogenesis in a dose-dependent fasion. Mechanically,Real-time PCR and Western blot analysis revealed the expressions both VEGF and Flk-1 significantly in- creased in CREG+/EBs.Moreover,after treatment of CREG+ /EBs with neurtralizing antibody against VEGF,the rapid vasculogenesis was significantly repressed.Conclusions Our data strongely demonstrate that CREG play a pivotal role in accelerating vasculogenesis in development of ES cells. VEGF,as its important downstream effector,mediated this bio-function.
