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

Figure 1 Schematic diagram of gene transfection. Transducer with contact gel (CG) was placed on the cell suspension covered with a 6-&mgr;m poly-ester film (PEF) under 1.9 MHz continuous ultrasound with a 80.0 mW/cm² output intensity. The 24-well plate was kept in a water bath at 37°C, and a sponge mat was placed in the water bath.

Figure 2 Photomicrographs show cultured human endothelial cells on d 7. The cells appeared to be a monolayer of very flat, polygonal-shaped cells. A: × 100; B: × 200.

Figure 3 Immunofluorescence study of cultured human endothelial cells. A: Shows immunofluorescence positive-stained cells treated with Factor VIII related antigen. The cells were stained brown (× 400); B: Shows positive-stained cells in vitro treated with DiI-Ac-LDL. More than 95% HUVECs are found to be red fluorescence (× 400).

Figure 4 Immunofluorescence study (× 400) of cultured human endothelial cells. A: Shows positive-stained human endothelial cells with KDR receptor; B: Shows negative-stained human hepatoma cells without KDR receptor.

Figure 5 Identification of recombinant pEGFP-KDR-TK by restriction enzyme digestion (SalI/XhoI) and electrophoresis. Lane 1: DNA markers (λDNA/HindIII); Lane 2-5: pEGFP-KDR-TK was digested with SalI/ XhoI.

Figure 6 Fluorescent microscopy showed EGFP expression 48 h after pEGFP-KDR-TK transfection. A: Shows no obvious EGFP expression in cells without microbubbles and ultrasound (× 200); B: 1.7% cells exposed to ultrasound only shows EGFP expression (× 200); C, D: Significant increase in EGFP expression in HUVECs exposed to both the ultrasound and SonoVue (C: × 200, D: × 400). About 20.3% HUVECs had EGFP expression.

Figure 7 HUVECs survival rate examined by trypan blue dye exclusion after transfection. Values are expressed as the average percentage of viable cells, n = 5 from 3 independent experiments. ^aP < 0.05 vs group A; ^cP > 0.05 vs group B.

Figure 8 In vitro cytotoxic effect of GCV on KDR-TK transfected HUVECs (KDR-TK/VEC), non-transfected HUVECs, and HepG2 cells. KDR-TK/VECs, non-transfected HUVECs, and HepG2 cells were cultured with various concentrations of GCV for 3 d and viability was estimated by MTT assay.