Killing effect of suicide gene system under control by KDR promotor on human umbilical vein endothelial cells.

OBJECTIVE

To study the killing effect of adenovirus-mediated double suicide gene under the regulation of kinase domain-containing receptor (KDR) promoter on human umbilical vein endothelial cells (HUVECs).

METHODS

The sequences of human KDR promoter gene, CD gene and TK gene were amplified by PCR, and the plasmid pKDR-CDglyTK was constructed. A two-step transformation protocol was employed for the construction of a recombinant adenoviral plasmid pAdKDR-CDglyTK that was transfected into 293 packaging cells to further multiply and purify the adenovirus. HUVECs were infected by the resultant recombinant adenovirus of different multiplicities of infection (MOI), and the infection rate was measured by observing the expression of green fluorescence protein (GFP). The infected cells were cultured in the culture media containing ganciclovir (GCV) and/or 5-fluorocytosine (5-FC) at different concentrations, and the killing effects were evaluated.

RESULTS

Recombinant adenovirus AdKDR-CDglyTK were successfully constructed, which could efficiently infect HUVEC cells, with the infection rate associated with the MOI of the recombinant adenovirus. HUVEC cells infected with AdKDR-CDglyTK were highly sensitive to the prodrugs, their survival rate correlated to both the concentration of the prodrugs and the MOI of the recombinant adenovirus. The killing effect of the two produrgs used in combination was much stronger than that of exclusive use of GCV or 5-FC.

CONCLUSIONS

Prodrug/KDR-CdglyTK system is effective in killing HUVEC cells, and its killing effect is correlated with the concentration of the prodrugs and the MOI of the recombinant adenovirus. Combination of the two prodrugs produces stronger killing effect on the cells.