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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。
Objective To construct the fusion expression vector pcDNA3.1-RI and to detect the relationship between ribonuclease inhibitor (RI) gene and angiogenin (ANG) and the effect on human umbilical vein endothelial cells HUVECs) proliferation and migration ability. Methods RI gene was amplified by RT-PCR and inserted into pcDNA3.1 after digestion. The fusion expression vector pcDNA3.1-RI was constructed and transfected into HUVECs by lipofectamine. RI and ANG genes were detected by RT-PCR The expression of RI, ANG, MMP-2 and MMP-9 were detected by Western blot. The interaction between ANG and RI was detected by CO-IP method. The proliferation activity of cells was detected by MTT assay. The cell cycle was detected by flow cytometry distributed. Results The eukaryotic expression plasmid was constructed successfully. The mRNA and protein expression of RI gene in pcDNA3.1-RI transfected group were significantly higher than those in control group (pcDNA3.1 empty vector group and untransfected plasmid group) (P <0.05), while the mRNA and protein expression of ANG gene decreased (P <0.05) and the expression of MMP-2 and MMP-9 protein decreased RI could be bound in the cells. After transfected with pcDNA3.1-RI plasmid into HUVECs, the proliferation activity of the cells was significantly decreased (P <0.05). The proportion of G0-G1 phase increased significantly and the S phase decreased. Conclusion The constructed eukaryotic expression plasmid can significantly increase the expression of RI gene and its protein. RI can directly reduce the expression of ANG at the transcriptional level and bind with ANG in the cell, thus affecting the proliferation and migration of endothelial cells.