Basic Research
Copyright ©2008 The WJG Press and Baishideng. All rights reserved.
World J Gastroenterol. Jan 14, 2008; 14(2): 224-230
Published online Jan 14, 2008. doi: 10.3748/wjg.14.224
Expression of thymidine kinase mediated by a novel non-viral delivery system under the control of vascular endothelial growth factor receptor 2 promoter selectively kills human umbilical vein endothelial cells
Ying Wang, Hui-Xiong Xu, Ming-De Lu, Qing Tang
Ying Wang, Hui-Xiong Xu, Qing Tang, Department of Medical Ultrasonics, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, Guangdong Province, China
Ming-De Lu, Department of Hepatobiliary Surgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, Guangdong Province, China
Correspondence to: Ming-De Lu, MD, DMSc, Department of Hepatobiliary Surgery, The First Affiliated Hospital, Sun Yat-Sen University, 58 Zhongshan Road II, Guangzhou 510080, Guangdong Province, China. lumdgd@yahoo.com.cn
Telephone: +86-20-87765183
Fax: +86-20-87765183
Received: June 12, 2007
Revised: October 26, 2007
Published online: January 14, 2008
Abstract

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.

Keywords: Microbubble; Ultrasound; Gene therapy; Vascular endothelial growth factor receptor 2; Human umbilical vein endothelial cells