| Objective:To study the chimeric promoter with high activity which is composed with CMV promoter and AFP enhancer;the specificity of the fused suicide gene CD/TK increases or not,which expressed in target cell by the regulation of fused promoter;and by magnetic cationic liposome with good biocompatibility as a carrier,suicide gene transfect into hepatoma cells,to observe the probability of enhancing target and safety of gene therapy.Methods:magnetic cationic liposomes were prepared by reverse phase evaporation,the expression vector of AFPE-pTK-IRES-CD as a fused suicide gene was construct by PCR,RT-PCR,fusion PCR, restriction enzyme and ligation etc.In vitro experiment was through transfect pAFPE-pTK-IRES-CD plasmid expression vector into HepG2 cell line using magnetic cationic liposomes as a vector.The transfection efficiency was detected by fluorescence microscope and flow cytometer, the express of CD/TK fused suicide gene in HepG2 cell was detected by RT-PCR and Western-blot.The toxicity of 5-FC and GCV on HepG2 cells was detected by MTT.The killing effect of suicide gene prodrug system on HepG2 cells expressed the suicide gene was detected by MTT and clone formation experiment.The effect of this system on the growth of HepG2 cells was observed by cell growth curve.The change of express level of the suicide gene before and after treatment was detected by immunohistochemistry and immunofluorescence.The change of transfection efficiency from prodrug 5-FC to 5-FU was detected by HPLC before and after treatment.Results:Magnetic cationic liposomes could conjugate with plasmid DNA as the complex,it absorbed on the cell membrane and went into cells,increased DNA quantities into cells and increased the efficiency of gene transfection.The complex could inhibit well the DNA degradation by the enzyme in blood serum and protect well the integrality of DNA molecule.When HepG2 cells were transfected after Green fluorescent protein(GFP) as a reporter gene combined with magnetic cationic liposomes,it found that it had higher transfection efficiency.5-FC and GCV in a high concentration(5-FC＞60μg/mL GCV＞0.6μg/ml) had some killing effect for HepG2 cells.For CD/TK+ HepG2 cells,when the 5-FC concentration was 40μg/ml and 80μg/ml,cell survival rates were 69.23% and 18.02%respectively.And for CD/TK+ HepG2 cells,under the condition of simultaneous existence for two prodrugs 5-FC and GCV, synergism effect was not found which reported by many studies,and the effect was lower than 5-FC alone.CD/TK+ HepG2 cells were treated 24h with 200μg/ml 5-FC,5-Fu concentration in cell supernatant was 168μg/ml,the transformation efficiency was 94%from 5-Fc to 5-Fu. Under 200μg/ml 5-FC,when pAFPE-pTK-IRES-CD plasmid had more 20%transfection efficiency,the relative survival rate of the cells decreased to 49.6%,this revealed that magnetic cationic liposomes-mediated suicide gene prodrug treatment had evident effect on tumor inhibition.Conclusions:Magnetic cationic liposomes can mediate well the transfection of suicide gene pAFPE-pTK-IRES-CD plasmid into HepG2 cells for,and have no evident toxicity and side effects,so it may be a new non-viral vector for gene therapy.5-FC and GCV have evident killing effect for CD/TK+ cells,but no synergism;and the effect is lower than that of 5-FC alone,but stronger than that of GCV.The fused suicide gene prodrug system has strong bystander killing effect,and as prodrug 5-FC＜200μg/ml,GCV＜50μg/ml,its toxicity is very low for HepG2 cells with no suicide gene.In vitro experiment reveals that magnetic cationic liposomes-mediated suicide gene under regulation by AFP has good targeted killing effect for tumor therapy.
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