The Selective Killing Effects Of TK Gene On Hepatocellular Carcinoma Cells Mediated By A Novel Targeted Gene Carrier

Objectives:1.Apolipoprotein E and cationic liposome were used to assemble a gene transport complex which can targetedly combine with hepatocyte or hepatocellular carcinoma cells,the transfection efficiency was detected.2.The apoE-lipoplexes can enclose pAFP-TK-IRES2-EGFP plasmid vector which contain the alpha-fetoprotein promoter,enhanced green fluorescent protein as a reporter gene and the herpes simplex virus thymidine kinase suicide gene,the complex was used to transfect liver cells or hepatocellular carcinoma cells,the specific expression levels of HSVtk were detected.3.The cytotoxic activities on liver cells or hepatocellular carcinoma mediated by the apoE-lipoplexes vector and the AFP promoter regulating HSVtk/ganciclovir suicide gene system were detected in vitro.Methods:1.Apolipoprotein E modified cationic liposome and normal cationic liposome were prepared respectively,plasmid pGenesil-1 containing EGFP report gene was carried and transfected into hepatocellular carcinoma cell lines HepG2,HUH-7 and Li-7,normal liver cell HL-7702 and colorectal cancer cell line SW480,transfection efficiencies were detected by flow cytometry.2.Detecting the stability of apoE-lipoplexes and pAFP-TK-IRES2-EGFP plasmid complexes under the DNase?digestion.3.The pAFP-TK-IRES2-EGFP plasmid carried by the apoE-lipoplexes was transfected into HepG2,HUH-7,Li-7,HL-7702 and SW480 cells respectively.RT-PCRand Western blotting methods were used to detect the mRNA and protein expression levels of HSVtk gene.4.With the pAFP-IRES2-EGFP plasmid as a control group,pAFP-TK-IRES2-EGFP was transfected into hepatocellular carcinoma cell lines HepG,HUH-7 and Li-7,after the prodrug ganciclovir was added,the cell death ratio was detected by flow cytometry.Results:1.The apoE-lipoplexes with a characteristic of targetedly binding to liver cells or hepatocellular carcinoma cells was successfully composed.Compared with the normal liposome,the transfection efficiency of apoE-lipoplexes was improved significantly,the difference was statistically significant(P<0.05).2.Afer pAFP-TK-IRES2-EGFP was transfected into HepG2,HUH-7,Li-7,HL-7702 and SW480 cells,RT-PCR and western blotting detection results showed that,HSVtk was expressed in AFP positive HepG2,HUH-7,Li-7 cells,but not expressed in AFP negative HL-7702 and SW480 cells.3.Compared with the control group,the death ratios of hepatocellular carcinoma cells HepG2,HUH-7 and Li-7 were significantly increased,the difference was statistically significant(P<0.05)Conclusions:1.The cationic liposome apoE-lipoplexes modified by apolipoprotein E has higher transfection efficiency on liver cells or hepatocellular carcinoma cells.2.ApoE-lipoplexes mediated suicide gene eukaryotic expression plasmid pAFP-TK-IRES2-EGFP was successfully expressed in alpha fetoprotein positive hepatocellular carcinoma cell lines HepG2,HUH-7 and Li-7.3.ApoE-lipoplexes mediated HSVtk/GCV suicide gene system has a killing effect on hepatocellular carcinoma cells HepG2,HUH-7 and Li-7,but not on normal liver cells.