改良内皮抑素对人脐静脉内皮细胞抑制作用的实验研究  

Inhibitory Effect of Modified Endostatin on Human Umbilical Vein Endothelial Cells

作　　者：肖楠[1] 田霈[1] 罗鑫[1] 齐东华[1] 倪双[1] 刘平[1] 

机构地区：[1]哈尔滨医科大学第一临床医学院眼科,黑龙江哈尔滨150001

出　　处：《现代生物医学进展》2012年第14期2632-2637,2641,共7页Progress in Modern Biomedicine

基　　金：黑龙江省自然科学基金项目(QC2010113)

摘　　要：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。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

关 键 词：改良内皮抑素(RGDRGD-ES) 内皮抑素(ES) 人脐静脉内皮细胞(HUVEC) 角膜新生血管(CNV) 

分 类 号：Q813[生物学—生物工程] R772.2[医药卫生—眼科]