肖楠 田霈 罗鑫 齐东华 倪双 刘平.改良内皮抑素对人脐静脉内皮细胞抑制作用的实验研究[J].现代生物医学进展英文版,2012,12(14):2632-2637. |
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 UmbilicalVein Endothelial Cells |
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DOI: |
中文关键词: 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) |
英文关键词: RGDRGD-endostatin (RGDRGD-ES) Endostatin (ES) Human umbilical vein endothelial cell (HUVEC) Corneal
neovascularization(CNV) |
基金项目:黑龙江省自然科学基金项目(QC2010113) |
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中文摘要: |
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(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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