摘要
AIM: To investigate the killing efficiency of a recombinant plasmid containing a thymidine kinase (TK) domain insert driven by the vascular endothelial growth factor receptor 2 (VEGFR2) promoter (KDR) on vascular endothelial cells.METHODS: The KDR-TK fragment was extracted from pBluescript Ⅱ KDR-TK plasmid by enzymatic digestion with Xho I and Sal I. The enhanced green fluorescence protein (EGFP) carrier was extracted from pEGFP by the same procedure. The KDR-TK was inserted into the pEGFP carrier to construct pEGFP-KDR-TK. Using ultrasound irradiation and microbubble, pEGFP-KDR-TK was transferred into human umbilical vein endothelial cells (HUVECs). The transient infection rate was estimated by green fluorescent protein (GFP) expression. Transfected HUVECs, non-transfected HUVECs, and HepG2 cells were cultured in the presence of different concentrations of ganciclovir (GCV), and the killing efficacy of HSV-TK/GCV was analyzed by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: The recombinant pEGFP-KDR-TK was successfully constructed by inserting the KDR-TK fragment into the pEGFP carrier. Transfected HUVECs showed cytoplasmic green fluorescence, and the transient transfection rate was about 20.3%. Pools of G418-resistant cells exhibited a higher sensitivity to theprodrug/GCV compared to non-transfected HUVECs or non-transfected HepG2 cells, respectively. CONCLUSION: KDR promoter and the suicide gene/prodrug system mediated by diagnostic ultrasound combined with microbubble can significantly kill HUVECs. Such therapy may present a novel and attractive approach to target gene therapy on tumor vessels.
AIM： To investigate the killing efficiency of a recombinant plasmid containing a thymidine kinase （TK） domain insert driven by the vascular endothelial growth factor receptor 2 （VEGFR2） promoter （KDR） on vascular endothelial cells. METHODS： The KDR-TK fragment was extracted from pBluescript Ⅱ KDR-TK plasmid by enzymatic digestion with XhoI and SalI. The enhanced green fluorescence protein （EGFP） carrier was extracted from pEGFP by the same procedure. The KDR-TK was inserted into the pEGFP carrier to construct pEGFP- KDR-TK. Using ultrasound irradiation and microbubble, pEGFP-KDR-TK was transferred into human umbilical vein endothelial cells （HUVECs）. The transient infection rate was estimated by green fluorescent protein （GFP） expression. Transfected HUVECs, non-transfected HUVECs, and HepG2 cells were cultured in the presence of different concentrations of ganciclovir （GCV）, and the killing efficacy of HSV-TK/GCV was analyzed by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide （MTT） assay. RESULTS： The recombinant pEGFP-KDR-TK was successfully constructed by inserting the KDR-TK fragment into the pEGFP carrier. Transfected HUVECs showed cytoplasmic green fluorescence, and the transient transfection rate was about 20.3%. Pools of G418-resistant cells exhibited a higher sensitivity to the prodrug/GCV compared to non-transfected HUVECs or non-transfected HepG2 cells, respectively. CONCLUSION： KDR promoter and the suicide gene/ prodrug system mediated by diagnostic ultrasound combined with microbubble can significantly kill HUVECs. Such therapy may present a novel and attractive approach to target gene therapy on tumor vessels.
基金
New Century Distinguished Scholar Supporting Program of Ministry of Education (80000-3171404)
The National Natural Science Foundation of China, No. 30300082, No. 30470467
