[Construction of VEGF recombinant plasmid pcDNA/V and its expression in model rats with acute myocardial ischemia].
The cDNA encoding human Vascular Endothelial Growth Factor 165 (VEGF165) was amplified using RT-PCR from human tonsil tissue and cloned into eukaryotic expression vector pcDNA3.1 (+). The recombinant plasmid pcDNA/V was transferred into 293 cells mediated by liposome and the cells stably expressing VEGF were selected under the pressure of G418. ELISA and Western blotting demonstrated that the eukaryotic expression vector pcDNA/V was successfully constructed and its corresponding protein could be expressed efficiently in vitro. Chick Charioallantoic Membrane (CAM) bioassay showed that recombinant protein has biological activity of hVEGF. Model rats with acute myocardial ischemia were used to further study the expression of VEGFin vivo. The model rats were divided randomly into three groups: control group, pcDNA3.1 (+) group and pcDNA/V group. 50microL naked plasmid DNA or saline was intramyocardially injected at three sites into the border zone of infarction. The hearts of rats were excised and fixed histologically, then the infarction sizes were studied by immunohistochemical staining and electron microscope after four weeks. Immunohistochemical staining for VEGF appeared to be negative in control and pcDNA3.1 (+) groups. In pcDNA/V group, myocardial cells in infarction border zone showed positive staining for VEGF in cytoplasm. Ultrastructural anaylsis showed that there were visible hyperplasia of vascular endothilium in pcDNA/V group. The control and pcDNA3.1 (+) groups showed less capillary hyperplasia. In this study, VEGF165 gene was successfully cloned and its protein expressed in vitro and in vivo was of bioactivity, which provides a basis for the further study of biological functions of human VEGF.
Wang
Ya-Mei
YM
Experiment Center, Capital University of Medical Sciences, Beijing 100054, China.
Liu
Bing
B
Sun
Li-Cui
LC
Yan
Yu-Dong
YD
Si
Yang
Y
Qi
Ya-Hui
YH
D000818:Animals; D002460:Cell Line; D002645:Chickens; D049033:Chorioallantoic Membrane / Q000098:blood supply; D004195:Disease Models, Animal; D015316:Genetic Therapy; D006801:Humans; D008297:Male; D009203:Myocardial Infarction / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D011897:Random Allocation; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000627:therapeutic use*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics*
D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A