Inhibitory Effect of Modified Endostatin on Human Umbilical Vein Endothelial Cells

Objective: To investigate the inhibitory effect of RGDRGD-ES on HUVEC in vitro.Methods: A modified human endostatin gene containing RGDRGD motif was obtained by rapid site-directed mutagenesis.The RGD mutated endostatin gene was expressed by a prokaryotic expression vector and purified by Ni-NTA resin.Automated gene sequencing and Western blot analysis were used to identify RGDRGD-ES gene and protein respectively.HUVEC cultured in vitro were exposed to RGDRGD-ES protein at different concentrations(0 μg/ml,10 μg/ml,20 μg/ml,30 μg/ml,40 μg/ml,50 μg/ml) and ES at 30 μg/ml for different time duration(24 h,48 h,72 h,96 h).Cell viabilities were monitored by 3,4,5dimethyliazol-2,5diphenyltetrazolium bromide(MTT) assay.The apoptosis rates at 24 h were detected by flow cytometric analysis.Results:Modified endostatin gene containing RGDRGD motif was confirmed by automated gene sequencing.The prokaryotic expression vector containing RGDRGD-ES was successfully constructed,and RGDRGD-ES expression was identified by Western blot.RGDRGD-ES clearly reduced HUVEC viability compared to control group(P0.01) and ES group(P0.01).RGDRGD-ES induced loss of cell viability by a dose dependant manner among 10 μg/ml,20 μg/ml,30 μg/ml groups(P0.01),while no significant differences were found in 30 μg/ml,40 μg/ml,50 μg/ml groups(P0.01).RGDRGD-ES also induced loss of cell viability by a time dependant manner within 72 h(P0.01),however,there was no significant difference between 72 h and 96 h(P0.05).Consistent with MTT assay,RGDRGD-ES induced HUVEC apoptosis by a dose dependant manner among 10 μg/ml,20 μg/ml,30 μg/ml groups(P0.01),while no significant differences were found in 40 μg/ml,50 μg/ml groups(P0.05).Conclusion:RGDRGD-ES can be obtained by rapid site-directed mutagenesis and prokaryotic expression vector.RGDRGD-ES inhibits HUVEC cell viability and induces cell apoptosis significantly.The optimal dose and time of RGDRGD-ES incubation are 30 μg/ml and 72 h.Modified endostatin with the RGDRGD motif is more effective than ES in inhibition of HUVEC cell viability and induction of HUVEC cell apoptosis.