title: Effect of recombinant plasmid pEGFP-AFP-hTNF on liver cancer cells (HepG2 Cells) in vitro when delivered by PEG-PEI/Fe₃O₄ nanomagnetic fluid.
abstract: OBJECTIVE
  Gene delivery into liver cancer cells has been a problem. This study aimed to understand the effect of using PEGI/Fe₃O₄ nanomagnetic fluid as a gene vector for liver cancer gene therapy. An AFP enhancer aids in the expression of tumor-specific foreign genes in AFP-producing cancer cells like HepG2 cells, and was utilized in the delivery method in this study.
  
  
  METHODS
  We constructed recombinant plasmid PEGFP-AFP-hTNFα, which was transfected into AFP positive HepG2 cells and AFP negative Hela cells by PEG-PEI/Fe₃O₄ nanomagnetic fluid. Fluorescence microscopy was used to evaluate the transfection rate of the hTNFα gene in the HepG2 cells 12 hours after transfection. Reverse transcription polymerase chain reaction (RT-PCR) and western blot were used to detect expression of hTNFα gene in the HepG2 cells 48 hours after transfection. Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the inhibitory effect of hTNFα on the proliferation of HepG2 cells. Flow cytometry was used to analyze the apoptosis of HepG2 cells.
  
  
  RESULTS
  Plasmid PEGFP-AFP-hTNFα delivered by PEG-PEI/Fe₃O₄ nanomagnetic fluid was successfully transfected into HepG2 cells and expressed in the HepG2 cells. The transfection efficacy of hTNFα gene delivered by PEG-PEI/Fe₃O₄ nanomagnetic fluid was superior to that of hTNFα gene delivered by lipofectamine in HepG2 cells. RT-PCR and western blot demonstrated that hTNFα gene was expressed in HepG2 cells that were transfected with complexes of nanomagnetic fluid/PEGFP-AFP-hTNFα. MTT and flow cytometry showed that the hTNFα gene markedly exerted a cell killing effect.
  
  
  CONCLUSIONS
  PEG-PEI/Fe₃O₄ nanomagnetic fluid successfully transfected PEGFP-AFP-hTNFα into HepG2 cells and induced expression of hTNFα gene in the HepG2 cells, thus showing promise as a gene vector for liver cancer gene therapy. Furthermore, an AFP enhancer can specifically increase the expression of target genes in cells positive for AFP.
authors.affiliation: Hepatobiliary Surgery of Xiangya Hospital, Central South University, Changsha, Hunan 41008, China.
authors.forename: Baoxiong
authors.initials: B
authors.lastname: Zhuang
authors.forename: Yangde
authors.initials: Y
authors.lastname: Zhang
authors.forename: Jian
authors.initials: J
authors.lastname: Peng
authors.forename: Haowei
authors.initials: H
authors.lastname: Zhang
authors.forename: Tiehui
authors.initials: T
authors.lastname: Hu
authors.forename: Jie
authors.initials: J
authors.lastname: Zeng
authors.forename: Yifan
authors.initials: Y
authors.lastname: Li
mesh_terms: D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000235:genetics; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000628:therapy*; D058185:Magnetite Nanoparticles / Q000008:administration & dosage*; D010957:Plasmids; D011092:Polyethylene Glycols / Q000008:administration & dosage*; D011094:Polyethyleneimine / Q000008:administration & dosage / Q000031:analogs & derivatives*; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*; D000509:alpha-Fetoproteins / Q000235:genetics*
chemical_list: D058185:Magnetite Nanoparticles; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D000509:alpha-Fetoproteins; C485184:enhanced green fluorescent protein; C544016:poly(ethylene glycol)-co-poly(ethyleneimine); D049452:Green Fluorescent Proteins; D011092:Polyethylene Glycols; D011094:Polyethyleneimine