| Objective:(1)Study on effects of DAG on HUVEC cells of proliferation.(2)Study on effects of DAG on HUVEC cells of migration and the lumen formed.(3)To assay the possible mechanism of proliferation of DAG on HUVEC cells by measuring the protein expression.(4)Zebrafish was used as a model to assay the effects of DAG on antiangiogenesis and its possible mechanism.Methods:(1)Study on the effects of DAG on HUVEC cells of proliferation was carried out in vitro and the cultured system of HUVEC cells was established.(1)HUVEC cells were detected by MTT methods in different concentrations of DAG.The proliferation inhibition rates and half inhibitory concentrations IC50 on the control group(without drug)in different time were determined.(2)The morphology of cells on different concentrations of DAG(0.00μg/ml,10.00μg/ml,20.00μg/ml,40.00μg/ml,80.00μg/ml,160μg/ml)after24h treatment was assayed by inverted microscope.(2)Study on effects of DAG on HUVEC cells of migration and the lumen formed.(1)Migration ability of cell was measured by scratch wound model in vitro and used Transwell to assay the chemotactic motility of HUVEC cells treated with different concentrations of DAG.(2)Formation of capillary tube-like structures by HUVEC cells with different concentrations of DAG was assessed in a matrigel-based assay.(3)Study on the mechanism of DAG on HUVEC cells of inhibiting angiogenesis.After HUVEC cells treated with different concentrations of DAG(0.00μg/ml,40.00μg/ml,80.00μg/ml and 160.00μg/ml)for 24 h,(1)RT-PCR was used to evaluate the level of VEGFA,VEGFR-2,FGF-2,MMP-2,MMP-9expression in HUVEC cells,(2)ELISA was used to evaluate the level of VEGF,FGF-2 expression in supernant fluid of HUVEC cells,(3)Immunohistochemistry was used to evaluate the level of VEGF,FGF-2expression in HUVEC cells,(4)Western Blot was used to evaluate the level of MMP-2 expression in HUVEC cells.(4)Zebrafish was used as a model to test the effect of anti-angiogenesis.(1)Vascular staining of NBT/BCIP was used to test the effcet of the SIVs of zebrafish,(2)RT-PCR was used to evaluate the level of VEGFAA,VEGFR-2,FGF-2,FGFR-2 expression in zebrafishes.Results:(1)MTT results indicated that HUVEC cells were inhibited significantly by DAG.(2)Scratch test and Transwell assay showed DAG induced the in vitro migration of HUVEC cells in a dose-dependent manner.(3)DAG exhibited inhibitory effct on tube formation.And treatment with various concentrations of DAG clearly inhibited the tube formation of HUVEC cells in a dose-dependent manner.(4)RT-PCR,ELISA,Immunohistochemistry and Western Blot results all showed that the angiogenesis factors which were evaluated decreased after being treated with DAG.(5)Vascular staining of NBT/BCIP results showed DAG exhibited inhibitory effct on the growth of the SIVs of zebrafish.And RT-PCR results showed that VEGFAA,VEGFR-2,FGF-2,FGFR-2 decreased in a dose-dependent manner after being treated with DAG.Conclutions:(1)DAG significantly inhibited the migration,tube formation,new vascular network formation.(2)The effects of the DAG on angiogenesis of HUVEC cells was measured by RT-PCR,ELISA,Immunohistochemistry and Western Blot,and sugested that DAG suppressed expression of VEGFA,VEGFR-2,FGF-2,MMP-2,MMP-9.(3)DAG exhibited inhibitory effct on the growth of the SIVs of zebrafish.And RT-PCR results showed that VEGFAA,VEGFR-2,FGF-2,FGFR-2 decreased in a dose-dependent manner after being treated with DAG. |
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