PNP/MePdR Suicide Gene System Mediated By Attenuated Salmonella Typhimurium For Pancreatic Cancer Therapy

ObjectiveTo develop Attenuated Salmonella typhimurium SL3261harboring recombinantexpressing vector of pEGFP-C1-PNP and detecting the expresion in pancreatic cancer cells.To evaluate the killing effect of the PNP/MePdR suicide gene system on the BxPc-3pancreatic cancer cells using Attenuated Salmonella typhimurium as a vector.Methods1. PNP gene was amplified by PCR, constructed a recombinant eukaryotic expressingplasmid pEGFP-C1-ePNP and identified by enzyme digesting,PCR,and sequencinganalysis. pEGFP-C1-PNP vector was electrotransfered to Attenuated Salmonellatyphimurium SL3261.Then detected the stability of the morphology and suface antigen ofAttenuated Salmonella typhimunium containing plasmid. Meanwhile the reconstructedSL3261was cocultured with BxPc-3cells to detect their invasion.Fluorescence microscopeobservasion and RT-PCR was exploited to observe GFP and PNP mRNA in the host cells.2. The Attenuated Salmonella typhimurium containing PNP Gene was cocultured withBxPc-3cells in vitro, the prodrug MePdR was added and the killing effect of MePdR toBxPc-3cells and the bystander effect was measured by MTT and FCM.Results1. It was proved that PNP gene segments were inserted into pEGFP-C1correctly andtransfered into Attenuated Salmonella typhimunium successfully.The transfected BxPc-3cells by infection of SL3261containing the vector were shown with GFP and the720bptarget expression. 2. The BxPc-3cells infected by SL3261/pEGFP-C1-PNP were sensitive to MePdR.IC50value was0.1mg/L after5-d treatment. With MePdR1mg/L the infected cells half diedon day three.As for the bystander effect, Most cells were killed by MePdR with only5%positive cells in mixed culture.ConclusionIt was successfully constructed Attenuated Salmonella typhimunium SL3261containing recombined eukaryotic expressing plasmid pEGFP-C1-PNP and expressedcorrectly in pancreatic cancer cells.The damaging effect of the PNP/MePdR suicide genesystem on the BxPc-3pancreatic cancer cells mediated by Attenuated Salmonellatyphimurium are significant as well as the bystander effect.