Study On The Killing Effects Of HSV-tk Gene Mediated By ApoE Modified Lipofectamine 2000 On Hepatocellular Carcinoma

Objectives:1.ApoE(apolipoprotein E)can be coupled with Lipofectamine 2000 to construct the gene carrier for specific transfection of hepatocyte and hepatoma carcinoma cells.2.The transfection specificity and transfection efficiency of ApoE-Lipofectamine2000 were detected in vitro and its cytotoxicity was also tested.3.In vivo inhibition test,the plasmid pcDNA3.1-pSurvivin-TK carried by ApoE-Lipofectamine 2000 was combined with ganciclovir(GCV)system to detect the targeted killing effects on nude mice hepatic tumor model.Methods:1.Lipofectamine 2000 modified by ApoE was prepared by coupling ApoE with Lipofectamine 2000.2.In the vitro cell experiments,plasmid binding assay was used to detect the binding ratio of the liposome and the plasmid;pGenesil-1 plasmid carried by liposome was transfected into HL-7702 cells,HepG2 cells,HEK293 T cells and SW480 cells,and the transfection efficiency was detected by fluorescence microscopy and flow cytometer;MTT assay was applied to detect the toxicity of liposome on HepG2 cells.3.In the vivo experiments,HepG2 cells were inoculated subcutaneously in the right armpit of nude mice to establish the transplanted hepatocellular carcinoma tumor model of nude mice.The tumor-bearing nude mice were randomly divided into three groups when the tumor grew to about 200 mm3;A group: control group;B group:Lipofectamine 2000 + pcDNA3.1-pSurvivin-TK + GCV treated group;C group:ApoE-Lipofectamine 2000 + pcDNA3.1-pSurvivin-TK + GCV treated group.The changes of tumor volume and tumor weight were observed before and after treatment and the anti-tumor rate was calculated;RT-PCR and Western blotting methods were applied to observe the expression of HSV-tk plasmid mediated by liposome in tumor tissues;HE staining and TUNEL methods were used to observe the apoptosis and necrosis of tumor after treatment.Results:1.Lipofectamine 2000 modified by ApoE carrier has been successfully prepared,plasmid binding assay demonstrated that the mass-to-volume ratio of plasmid with Lipofectamine 2000 and ApoE-Lipofectamine 2000 were 1:2 and 1:2.5,respectively,the plasmid could be fully encapsulated.2.Flow cytometry test indicated that the transfection efficiency of ApoE-Lipofectamine 2000 on HL-7702 cells and HepG2 cells was higher than that of Lipofectamine 2000.The transfection efficiency was markedly different and was statistically significant(P < 0.05,P = 0.03,P = 0.001).In HEK293 T and SW480 cells,there was no significant difference in the transfection efficiency of the two liposomes(P > 0.05,P = 0.93,P = 0.55).3.MTT cytotoxicity assay showed that ApoE-Lipofectamine 2000 did not increase the toxic effect on HepG2 cells compared with that of Lipofectamine 2000(P > 0.05).4.The nude mice xenograft tumor model was established successfully.After treatment,the tumor volume and tumor weight were decreased compared with the control group.The tumor inhibition rates of B and C group were(29.46±2.51)% and(46.18 ± 1.79)%,respectively,and the difference was statistically significant(P < 0.05,P = 0.007).5.The results of Western blotting and RT-PCR showed that there was no obvious band in A group,while B and C groups could detect the expression of HSV-tkplasmid.6.HE staining and TUNEL assay showed that HSV-tk plasmid mediated by two liposomes had certain killing effects on tumor when combining with GCV treatment,but the inhibition effects of Lipofectamine 2000 mediated by ApoE was more obvious.Conclusions:1.A highly efficient hepatocellular carcinoma targeted gene delivery vector,ApoE-Lipofectamine 2000,was successfully constructed.2.Lipofectamine 2000 modified by ApoE has high affinity to hepatocyte and hepatoma carcinoma cells,and can be used to target the hepatocyte and hepatoma carcinoma cells.3.ApoE-Lipofectamine 2000 + pcDNA3.1-pSurvivin-TK + GCV system had the targeted killing effects on liver cancer.