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血管内皮细胞生长因子真核表达质粒的构建及其在心肌细胞中的表达(PDF)

《心脏杂志》[ISSN:1009-7236/CN:61-1268/R]

期数:
2003年第3期
页码:
218-222
栏目:
实验研究
出版日期:
2003-05-01

文章信息/Info

Title:
Construction of eukaryotic expression plasmid of VEGF165 gene and its expression in myocardial cells
作者:
易甫; 吕安林; 贾国良; 张荣庆
第四军医大学西京医院心内科,陕西西安710032
Author(s):
YI Fu; Lv An-lin; JIA Guo-liang; ZHANG Rong-qing
Department of Cardiology,Xi jing Hospital,Fourth Military Medical University,Xi'an,Shaanxi 710032,China
关键词:
血管内皮细胞生长因子; 真核表达载体; 心肌细胞
Keywords:
vascular endothelial growth factor; eukaryotic expression vector; myocardial cell
分类号:
Q58
DOI:
-
文献标识码:
A
摘要:
目的:构建血管内皮细胞生长因子(VEGF165)真核表达质粒pcDNA3.1(-)/hVEGF165,并通过转染心肌细胞来验证其生物活性。方法:应用DNA重组方法,将编码hVEGF165全长cDNA克隆于真核表达载体pcDNA3.1(-)中,构建pcDNA3.1(-)/hVEGF165真核表达质粒;应用阳性脂质体介导的基因转染技术,将真核表达质粒pcDNA3.1(-)/hVEGF165瞬时转染培养的大鼠心肌细胞中;采用RT-PCR,ELISA,Westernblot及免疫组化染色等方法检测VEGF165基因在心肌细胞中的表达情况;应用MTT法检测转染后细胞上清液中VEGF165的生物活性。结果:将构建好的真核表达质粒pcDNA3.1(-)/hVEGF165转染心肌细胞后,VEGFmRNA及蛋白表达水平明显增高,转染后的心肌细胞培养上清液具有促使内皮细胞增殖的生物活性。结论:成功构建了真核表达质粒pcDNA3.1(-)/hVEGF165,其转染心肌细胞后可获得较高水平VEGF蛋白的表达,所表达出VEGF蛋白具有生物学活性。
Abstract:
AIM:To construct a eukaryotic expression vector of human vascular endothelial growth factor (VEGF165) gene,and to investigate the transfection and expression of pc DNA3.1(-)/ hVEGF165 eukaryotic expression plasmid in myocardialcells.METHODS:pcDNA3.1(-)/ hVEGF 165 eukaryotic expression plasmid was constructed. Primarily cultured rat myocardial cells were transiently transfected with Lipofect AMINE2000.RT-PCR,ELISA,Western blot and immunohistochemical methods were used to detect the expression of VEGF gene and MTT to detect the biological activity of the conditioned medium after the transfection. RESULTS: There were significant increases of VEGF mRNA and protein in the myocardial cells transfected with pcDNA3.1(-)/hVEGF165.The conditioned medium after the transfection showed the biological activity to stimulate the proliferation of endothelial cells. CONCLUSION:The pcDNA3.1(-)/VEGF165,a eukaryotic expression plasmid for h VEGF165 gene is constructed. High levels of VEGF mRNA and protein expression can be obtained in the myocardial cells transfected with pcDNA3.1(-)/hVEGF165 eukaryotic expression plamid.The expressed protein has the biological activity.

参考文献/References

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备注/Memo

备注/Memo:
收稿日期：2002-8-29.
更新日期/Last Update: 2003-05-01