摘要
目的构建携带人血管内皮细胞生长因子165（VEGF165）基因的新型真核表达载体pIRES-EGFP-VEGF165，并通过转染人脐静脉内皮细胞株EA．hy926细胞，观察载体的体外表达和对血管内皮细胞增殖的影响。方法将VEGF165目的基因片段插入到真核表达载体pIRES-EGFP质粒中，按每孔加入质粒DNA1．2p,g介导体外瞬时转染人脐静脉内皮细胞株EA．hy926细胞，通过荧光实时定量逆转录一聚合酶链反应（RT—PCR，n=3）和Westernblot（n=5）分别检测mRNA和蛋白水平的表达，并通过MTT法（n=5）检测转染pIRES—EGFP—VEGFl65质粒对血管内皮细胞增殖的促进作用。结果成功构建pIRES—EGFP—VEGFl65真核表达载体，测序结果与预期相符；荧光计数显示体外瞬时转染人脐静脉内皮细胞株EA．hy926细胞的转染效率约为（11．2±2．5）％；荧光实时定量RT—PCR和Westemblot证实转染pIRES—EGFP．VEGFl65质粒组细胞在mRNA和蛋白水平表达的VEGF165均高于对照组（P〈0．01），MTT法检测表明转染pIRES-EGFP—VEGF165质粒组细胞增殖速度较对照组明显增加（P〈0．01）。结论成功构建pIRES—EGFP—VEGFl65真核表达载体，并实现其在人脐静脉内皮细胞株EA．hy926细胞中的表达，而且证实可以促进血管内皮细胞的增殖。
Objective To construct a recombinant eukaryotic expression vector pIRES-VEGF165- tPA containing functional region of human vascular endothelial growth factor 165 （VEGF165） gene, and investigate its in vitro expression and effect on the proliferation of vascular endothelial cells in transfected human umbilical vein endothelial cell lines （EA. hy926）. Methods The VEGF165 gene was inserted into the eukaryotic expression vector pIRES-EGFP to construct the recombinant plasmid, which was transfected into human umbilical vein endothelial cell lines （ EA. hy926） at 1.2 ~g/well subsequently. The transcrip- tion and expression of VEGF165 gene were detected by reverse transcription-polymerase chain reaction （ RT-PCR, n = 3 ） and Western blotting （ n = 5 ） respectively. And the effect on the proliferation of vascular endothelial cells was evaluated by MTI＂ assay （n = 5 ）. Results The sequence of pIRES-EGFP-VEGF165 approved that the gene of VEGF165 was inserted into the eukaryotic expression vector correctly. The trans- fection efficiency was about （ 11.2± 2. 5 ） %, detected by counting the positive cells of green fluorescence. The mRNA and protein levels of VEGF165 in the pIRES-EGFP-VEGF165 transfected group were signifi- candy higher than those in the control groups respectively （P 〈 0.01 ）. Also, MTT assay indicated the pro- liferation of endothelia ceils was greatly improved in the pIRES-EGFP-VEGF165 transfected group. Conclusion The recombinant eukaryotic expression vector pIRES-EGFP-VEGF165 is successfully constructed and can be expressed in transfected EA. hy926 cells, and it is proved that transfection with this vector could improve the proliferation of vascular endothelial cells.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2011年第4期499-502,共4页
Chinese Journal of Experimental Surgery
基金
基金项目：国家自然科学基金资助项目（30872543）
关键词
血管内皮细胞生长因子
真核表达载体
细胞增殖
内皮化
Vascular endothelial growth factor
Eukaryotic expression vector
Cellular proliferation
Endothelialization
