ABSTRACT
Objective To investigate the cytotoxicity of vascular endothelial growth factor (VEGF) siRNA combined with fusion suicide gene yCDglyTK on human gastric cancer cell line in vitro.MethodsThe gastric cancer cell line SGC7901 was transfeeted with blank plasmid pcDNA3.1 (-) null [pcDNA3.1 (-) group], or VEGF-siRNA expression plasmid pGenesil-shVEGF (SGC7901/shVEGF group),or fusion suicide gene plasmid pcDNA3.1 (-)CV-yCDglyTK (SGC7901/CDTK group),or combined gene plasmid pcDNA3.1 (-)shVEGF-yCDglyTK (SGC7901//shVEGF-CDTK group) with calcium phosphate nanoparticles (CPNPs).Un-transfected gastric cells were set as control group.The stable transfected cells were selected by G418.The target gene expression was verified by RT-PCR and Western-blot.After given prodrug 5-fluorocytosine (5-FC), the biologic characters variation, apoptotic morphology and apoptotic rate of cells in each group were observed through cell growth curve by MTT assays, by-stander effect, Hoechst 33258 staining and flow cytometry.The data was analyzed with SPSS 13.0 software and multiple groups’ comparison was analyzed with LSD test.ResultsFour gastric cancer cells lines transfected with different plasmids were successfully established.The expression of gene yCDglyTK was detected both in SGC7901/CDTK cells and SGC7901/shVEGF-CDTK cells.By MTT assays, the cell growth curve indicated that the A570 value of SGC7901/shVEGF cells, SGC7901/CDTK cells and SGC7901/shVEGF-CDTK cells decreased significantly compared with that of SGC7901 and SGC7901/null cells after a 24-hour 5-FC treatment (P＜0.01).When the percentage of stable gene trasfected SGC7901 cells was 60％, 80％and 100％, the cell relative viability was 13.09％±2.40％, 9.74％±2.83％ and 5.68％±1.03％,respectively. A large number of cells in SGC7901/CDTK and SGC7901/shVEGF-CDTK group appeared typical apoptotic morphology under fluorescence microscope.The result of flow cytometry showed that the apoptosis rates in SGC7901/shVEG group、 SGC7901/CDTK group and SGC7901/shVEGF-CDTK group were 16.40％ ±4.68％, 57.63％ ± 4.96％ and 69.07％ ± 4.69％,respectively, and there were significant differences compared with control (P＜0.01).Conclusion VEGF siRNA combined with suicide gene can effectively kill gastric cancer cell line SGC7901.Apoptosis induction may be one of the important mechanisms of killing tumor cells.