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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（10 μg/ml、20 μg/ml、30 μg/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30 μg／ml 与40 μg／ml 、50 μg／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（10 μg/ml、20 μg/ml、30 μg/ml）依赖性(P＜ 0.01)，30 μg／ml 与40 μg／ml、50 μg／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30 μg／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。

英文摘要:

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.

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