| Objective:1.To identify the possibility of using nano-magnetic fluids as a gene vector for liver cancer gene therapy.2.To study recombinant plasmid pEGFP-AFP-TK delivered by nano-magnetic fluds targeting killed liver cancer cells of AFP expression(HepG2 cells) in vitroMethods:Constructed recombinant plasmid pEGFP-AFP-TK which was delivered by nano-magnetic fuilds into AFP positive HepG2 cells and AFP negative SMMC-7721.The recombinant plasmid pEGFP-AFP-TK contain enhanced green fluorescent protein(EGFP),AFP enhancer and TK gene.The fluorescence was detected in order to evaluate the transfection rate after transfecting the plasmid into the HepG2 cells in 20h,40h,60h.RT-PCR and Western-blot were used to detect expression of TK gene after transfection in 40h.MTT assay was used to evaluate the effect of TK on the proliferation and bystander effect of HepG2 cells in ganciclovir(GCV).Flow cytometry was used to analysis the anpoptosis of HepG2 cells treating with the ganciclovir(GCV).Results:Nano-magnetic fluids have able to delivered the plasmid pEGFP-AFP-TK into HepG2 cells and TK gene was successfully expressed.The transfection efficacy of nano-magnetic fluids was superior than Lipofectamine in HepG2 cells.The AFP enhancer make the plasmid targeted in the AFP expressed HepG2 ceils.RT-PCR and Western-blot demonstrated that TK gene was expressed in HepG2 cells after being transfected with nano-magnetic fluids/pEGFP-AFP-TK.MTT and flow cytometry showed TK gene exerts cell-killing and bystander effect in the drug of GCV.Conclusions:1 Nano-magnetic fluids could successfully deliver pEGFP-AFP-TK into HepG2 cells and induce expression of TKgene,it is promising gene vector for liver cancer gene therapy.2 AFP enhancer can specifically enhance the expression of target gene within the cell with positive AFP.The HepG2 cell with positive AFP has killing effect and bystander effect with GCV,not to the SMMC7721 cell with negative AFP...
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