[Recombinant plasmid pEGFP-AFP-TK delivered by nano-magnetic fluids targeting killed AFP positive HepG2 cells in vitro].
OBJECTIVE
To study recombinant plasmid pEGFP-AFP-TK delivered by nano-magnetic fluids targeting killed AFP positive HepG2 cells in vitro.


METHODS
Constructed recombinant plasmid pEGFP-AFP-TK which was delivered by nano-magnetic fluids into AFP positive HepG2 cells and AFP negative SMMC-7721.The fluorescence was detected in order to evaluate the transfection rate, and RT-PCR and Western blot were used to detect the expression of TK gene after transfection. MTT assay was used to evaluate the effect of TK on the proliferation and bystander effect of HepG2 cells in ganciclovir (GCV). Flow cytometry was used to analysis the apoptosis of HepG2 cells.


RESULTS
Nano-magnetic fluids delivered plasmid pEGFP-AFP-TK into HepG2 cells and TK gene was successfully expressed, and the transfection efficacy of nano-magnetic fluids was superior to that of Lipofectamine. RT-PCR and Western blot results demonstrated that TK gene was expressed in HepG2 cells after being transfected with nano-magnetic fluids/pEGFP-AFP-TK. MTT and flow cytometry results showed TK gene exerts cell-killing and bystander effect.


CONCLUSIONS
Nano-magnetic fluids could successfully deliver pEGFP-AFP-TK into HepG2 cells and induce expression of TK gene, which is promising gene vector for liver cancer gene therapy. AFP enhancer can specifically enhance the expression of target TK gene within the AFP positive cell, and induce bystander effect and apoptosis in the AFP positive HepG2 cell with with GCV.
National Key Laboratory of Nanobiological Technology, Ministry of Health, Changsha, China. fordluo@yahoo.com.cn
Yong
Y
Luo
Yang-de
YD
Zhang
Jian
J
Peng
Huan-xin
HX
Yan
Dun
D
Xue
Kai
K
Wu
Lin
L
Zhou
Shi-wei
SW
Hang
Jin-feng
JF
Zhao
D000998:Antiviral Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D024201:Bystander Effect; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology / Q000628:therapy; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005434:Flow Cytometry; D015774:Ganciclovir / Q000494:pharmacology*; D015316:Genetic Therapy / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology / Q000628:therapy; D008280:Magnetics; D008856:Microscopy, Fluorescence; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000295:instrumentation / Q000379:methods; D000509:alpha-Fetoproteins / Q000235:genetics / Q000378:metabolism*
D000998:Antiviral Agents; D011993:Recombinant Fusion Proteins; D000509:alpha-Fetoproteins; D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase; D015774:Ganciclovir