[1]李红彦,潘湘阳,姚雪,等.RI基因真核表达载体的构建及其对人脐静脉内皮细胞的影响[J].陆军军医大学学报(原第三军医大学学报),2012,34(16):1591-1595.
 Li Hongyan,Pan Xiangyang,Yao Xue,et al.Construction of eukaryotic expression vector pcDNA3.1-RI and its effect on human umbilical vein endothelial cells[J].J Amry Med Univ (J Third Mil Med Univ),2012,34(16):1591-1595.
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RI基因真核表达载体的构建及其对人脐静脉内皮细胞的影响(/HTML )
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陆军军医大学学报(原第三军医大学学报)[ISSN:1000-5404/CN:51-1095/R]

卷:
34卷
期数:
2012年第16期
页码:
1591-1595
栏目:
论著
出版日期:
2012-08-30

文章信息/Info

Title:
Construction of eukaryotic expression vector pcDNA3.1-RI and its effect on human umbilical vein endothelial cells
作者:
李红彦潘湘阳姚雪熊东梅陈俊霞
重庆医科大学基础医学院细胞生物学与遗传学教研室,分子医学与肿瘤研究中心
Author(s):
Li Hongyan Pan Xiangyang Yao Xue Xiong Dongmei Chen Junxia
Department of Cell Biology and Genetics, Molecular Medicine and Cancer Research Center, College of Basic Medical Sciences, Chongqing Medical University, Chongqing, 400016, China
关键词:
核糖核酸酶抑制因子人脐静脉内皮细胞血管生成素真核表达
Keywords:
ribonuclease inhibitor human umbilical vein endothelial cells angiogenin eukaryotic expression
分类号:
R322.12;R329.23;R394-33
文献标志码:
A
摘要:
目的      构建融合表达载体pcDNA3.1-RI,并检测核糖核酸酶抑制因子(ribonuclease inhibitor,RI)基因与血管生成素(angiogenin, ANG)的关系及对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)增殖及迁移能力的影响。      方法      用RT-PCR方法扩增RI基因,酶切后将其插入pcDNA3.1,构建融合表达载体pcDNA3.1-RI,在脂质体介导下转染HUVECs,RT-PCR检测RI、ANG基因的mRNA表达水平;Western blot检测RI、ANG、MMP-2、MMP-9的表达水平;CO-IP法检测ANG和RI的相互作用,MTT法检测细胞的增殖活力,流式细胞仪检测细胞周期分布。      结果      真核表达质粒构建成功;转染pcDNA3.1-RI组细胞RI基因的mRNA及蛋白的表达较2个对照组(转染pcDNA3.1空载体组和未转染质粒组)均呈显著性增加(P<0.05),而ANG基因的mRNA及蛋白的表达均降低(P<0.05),MMP-2、MMP-9蛋白表达水平亦降低(P<0.05);CO-IP法检测到ANG和RI在细胞内能结合;转染pcDNA3.1-RI质粒到HUVECs细胞后细胞的增殖活力明显降低(P<0.05),G0~G1期比例明显增加,S期减少。      结论      成功构建的真核表达质粒能显著增加RI基因及其蛋白水平的表达,RI可以直接在转录水平上降低ANG的表达,在细胞内与ANG结合,从而影响内皮细胞的增殖、迁移能力。
Abstract:
Objective      To construct an eukaryotic expression vector of ribonuclease inhibitor (RI) gene, pcDNA3.1-RI, and to investigate the effect of pcDNA3.1-RI on the growth of human umbilical vein endothelial cells (HUVECs) and the relationship between RI and angiogenin (ANG).       Methods      RI gene segments amplified by RT-PCR and digested by restriction enzymes were inserted into pcDNA3.1 to construct the recombinant vector pcDNA3.1-RI, which was transferred into HUVECs by liposomes. The mRNA levels of RI and ANG were detected by RT-PCR, and the protein expression levels of RI, ANG, MMP-2 and MMP-9 were detected by Western blotting. The interaction between ANG and RI was examined by co-immunoprecipitation (Co-IP). The proliferation activity of HUVECs was detected by MTT assay, and cell cycle distribution was detected by flow cytometry.       Results      The recombinant vector pcDNA3.1-RI was successfully constructed. The mRNA and protein expression of RI in the experimental group (HUVECs transfected with pcDNA3.1-RI) significantly increased as compared with that in the control group (HUVECs transfected with pcDNA3.1) and in the blank control group (P<0.05). The mRNA and protein expression of ANG decreased in the experimental group, and MMP-2 and MMP-9 protein expression decreased as well (P<0.05). In the experimental group, cell proliferation activity of HUVECs significantly decreased (P<0.05), and the cells were arrested at G1-S phase. The Co-IP detected the interaction between ANG and RI.       Conclusion      Eukaryotic expression vector pcDNA3.1-RI is constructed successfully, and it can increase the mRNA and protein expression of RI. RI can interact with ANG and downregulate ANG expression at the transcription level to inhibit the proliferation and migration of HUVECs.

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更新日期/Last Update: 2012-07-29