Research On Construction Of The PEGFP-AFP-TK, And It's Transfection Into Hepatoma Cells By PEG-PEI/Fe₃O₄ Nano-magnetic Fluids.

Objective:This study was to construct a recombinant eukaryotic expression vector containingα-fetoprotein(AFP)promoter-mediated HSV/TK suicide gene,and analyze its specific expression on AFP-positive hepatoma cells.And to identify the possibility of using PEG-PEI/Fe₃O₄ nano-scale magnetic fluids as gene vector for liver cancer gene therapy.Methods:The promoter of AFP gene,HSV-TK gene were amplified by PCR.The gene segments mentioned above were cloned into the multiple cloning sites of the eukaryotic expression vector pEGFP-N1, pEGFP-AFP-TK,was constructed.Then it was transfected into AFP-positive HepG2 and AFP-negative SMMC7721 hepatoma cells by PEG-PEI/Fe₃O₄ nano-magnetic fluids.Finally,the expression of the target gene in AFP-positive HepG2 and AFP-negative SMMC7721 hepatoma cells was detected by EGFP and Western blot.MTT assay was used to evaluate the effect of HSV/TK on the proliferation of HEPG2 cells.Results:The promoter of AFP gene,HSV-TK gene were amplified by PCR(about 247bp,1159bp,1381bp),and connected.AFP and TK genes were proved to be correct by DNA sequence analysis.There is no difference between these fragments sizes with prolepsis.AFP-promoter could drive interesting gene target expression in AFP positive cells (HEPG2),and there was no expression in AFP negative cells(SMCC). The transfection efficacy of PEG-PEI/Fe₃O₄ nano-magnetic fluids was superior than lipofectamine.The growth of hepatoma HepG2 cells have a certain inhibition after the suicide gene transferred.Conclusions:Hepatocellular carcinoma-specific eukaryotic expression vector carrying AFP promoter and HSV/TK suicide gene, pEGFP-AFP-TK,was sucessfully constructed.Using EGFP and Western blot method,AFP promoter driven HSV /TK gene system could be specifically expressed in AFP-positive hepatoma HepG2 cells.The transfection efficacy of PEG-PEI/Fe₃O₄ nano-magnetic fluids was superior than lipofectamine in HEPG2 cells.With MTT detected that the growth of hepatoma HepG2 cells have a certain inhibition after the suicide gene transferred.This experiment lays a solid foundation for the future research.