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Construction of conditionally replicationcompetent adenoviral vectors targeting hepatocarcinoma and their in vitro antitumor effect
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Abstract:
Objective: To construct a novel conditional duplicate adenovirus (CRA) regulated by AFP enhancer, AFP promoter and TK suicide gene, and to observe its duplicate abilities, cytopathic effects and cytotoxity against hepatocarcinoma cells when combined with predrug GCV.Methods: AFP gene promoter and enhancer were amplified using PCR from the genome DNA of HepG2 cells; pAFPpEGFPluc and pAFPepEGFPluc plasmids were constructed; pDC311AFPepE1A/CMVTK shuttle plasmid expression E1A gene was then constructed under the control of AFP promoter, AFP enhancer (AFPep) and TK gene. Ad.AFPepE1A/CMVTK vector was constructed by AdMax system. ElA expression, cytopathic effects and cytotoxity against hepatocarcinoma cells of Ad.AFPepE1A/CMVTK were evaluated by Western blotting, flow cytometry, CCK8 assay, respectively. Results: Ad.AFPepE1A/CMVTK was successfully constructed, which could selectively replicate in AFP positive HepG2 cells and specifically inhibit proliferation of AFP positive cells. Survival rates of AFP positive HepG2 and Hep3B cells after treated with combination of Ad.AFPepE1A/CMVTK and predrug GCV were (10.35±1.07)% and (15.49±5.80)%, respectively, which were significantly lower than those of AFP negative Chang liver cells and lung cancer NCIH460 cells (\[73.55±4.36\]% and \[74.54±9.89\]%, respectively, P<0.01). Conclusion: The constructed Ad.AFPepE1A/CMVTK, when combines with TK/GCV suicide gene system, can specifically kill AFP positive hepatocarcinoma cells, which may have a future in genebased therapy against hepatocarcinoma.
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Project Supported:
Supported by the Key Basic Research Program (973) of China (No.2004CB518804); the National Natural Science Funds for Distinguished Young Scholar (No.30325043)
Clc Number:
