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Archive > Volume 17 Issue 3 > 2010,17(3):297-301. DOI:10.3872/j.issn.1007-385X.2010.3.010 Prev Next

Basic Research

pAFPP53EGFP plasmid containing AFP regulation sequence induces apoptosis of AFP positive hepatoma cells

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    Abstract:
    Objective:To observe the targeting proapoptotic effect of expression vector containing AFPregulation sequence on AFP positive hepatoma cells. Methods: AFP promoter, silencer and the most remote enhancer Ⅲ were ligated to construct a 12 kb AFP regulation sequence, which was then used to construct a pAFPEGFP plasmid. The pAFPEGFP was used to transfect human hepatoma HepG2 (AFP positive), human hepatoma SMMC7721 (AFP negative) and human cervical carcinoma HeLa (AFP negative) cells; the fluorescent protein expression intensities were observed under fluorescence microscope. A pAFPP53EGFP plasmid was further constructed by inserting P53 gene into pAFPEGFP, which was then transfected into HepG2, SMMC7721 and HeLa cells. P53 protein expressions were detected by Western blotting analysis in different groups; apoptosis rates and cell cycles were examined by flow cytometry. Results: pAFPEGFP and pAFPP53EGFP recombinant plasmids were successfully constructed. The expression of EGFP fluorescent protein in pAFPEGFPtransfected AFP positive HepG2 cells was significant higher than those in AFP negative SMMC7721 and HeLa cells; P53 protein expression in HepG2 cells transfected with pAFPP53EGFP was also significantly higher than those in SMMC7721 and HeLa cells. The G1 phase proportion and apoptosis rate of pAFPP53EGFPtransfected HepG2 cells were significantly higher than those of SMMC7721 and HeLa cells (\[66.7±0.25\]% vs \[50.5±0.18\]%, \[51.0±020\]%, P<0.05; \[2.65±0.08\]% vs \[0.42±0.03\]%, \[0.39±0.02\]%, P<0.05), but S phase proportion of pAFPP53EGFPtransfected HepG2 cells was significantly lower than those of SMMC7721 and HeLa cells (\[20.1±022\]% vs \[29.8±0.18\]%, \[37.8±0.21\]%, P<0.05). Conclusion: The pAFPP53EGFP plasmid containing AFP regulation sequence can specifically target AFP positive hepatoma cells, inducing cell cycle arrest and apoptosis of hepatoma cells.

    Keywords:
    AFP gene ;  hepatoma cell ;  expression and regulation ;  P53 ;  cell cycle arrest ;  apoptosis

    Project Supported:
    Project supported by the Key Program of Science and Technology Supporting Foundation of Hebei Province (No.09276426D)

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