改良内皮抑素对人脐静脉内皮细胞抑制作用的实验研究

目的:探讨改良内皮抑素(RGDRGD-ES)对人脐静脉内皮细胞(HUVEC)的抑制作用,摸索RGDRGD-ES对HUVEC细胞抑制作用的相对最佳作用浓度和时间。方法:通过快速定点诱变PCR方法获得含有RGDRGD膜序的改良人内皮抑素基因,并构建其原核表达载体。表达、纯化改良内皮抑素(RGDRGD-ES),运用MTT法和流式细胞仪检测RGDRGD-ES对人脐静脉内皮细胞的抑制作用。结果:1.诱变了ES基因,获得了改良的RGDRGD-ES基因,并成功构建其原核表达载体。2.获得了RGDRGD-ES蛋白。3.改良的RGDRGD-ES能够有效抑制人脐静脉内皮细胞的生长(P<0.01);抑制率随着药物浓度(10μg/ml、20μg/ml、30μg/ml)的增加和作用时间(24 h、48 h、72 h)的延长而逐渐增加,具有浓度和时间依赖性(P<0.01);而30μg/ml与40μg/ml、50μg/ml组间、72 h与96 h组间无明显差异(P>0.05)。4.细胞凋亡率(作用24 h)具有药物浓度(10μg/ml、20μg/ml、30μg/ml)依赖性(P<0.01),30μg/ml与40μg/ml、50μg/ml组间凋亡率无明显差异(P>0.05)。结论:成功构建了改良RGDRGD-ES基因的原核表达载体,RGDRGD-ES蛋白在30μg/ml浓度作用72小时条件下能够有效抑制人脐静脉内皮细胞的生长,改良内皮抑素(RGDRGD-ES)对HUVEC的抑制作用较ES明显提高。

Inhibitory Effect of Modified Endostatin on Human UmbilicalVein 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(P<0.01) and ES group(P<0.01).RGDRGD-ES induced loss of cell viability by a dose dependant manner among 10 μg/ml,20 μg/ml,30 μg/ml groups(P<0.01),while no significant differences were found in 30 μg/ml,40 μg/ml,50 μg/ml groups(P<0.01).RGDRGD-ES also induced loss of cell viability by a time dependant manner within 72 h(P<0.01),however,there was no significant difference between 72 h and 96 h(P>0.05).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(P<0.01),while no significant differences were found in 40 μg/ml,50 μg/ml groups(P>0.05).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.