ABSTRACT
Objective To construct a new replicating adenovirus vector CNHK600-p53 carrying anti-tumor gene p53 and investigate its effect on hepatocarcinoma cell line PLC/PRF5. Methods The methylthiazolyl tetrazolium assay (MTT) was used to observe the killing effect of Fluorouracil, Mitomycin and Epirubicin alone or in combination with adenovirus CNHK600-p53 on PLC/PRF5. Results When 5-Fu at concentration of 400?g/ml, the inhibition rate was (65?4. 2) % ; with MMC at 1?g/ml, the rate was (41?1.9)%; and it was (65?1.8)% when EPI at concentration of 10?g/ml. With PLC/PRF5 infected by adenovirus CNHK600-p53 ( MOI = 0. 625) , the inhibition rate was significantly increased to (89?5. 3)%,(60?2.3)% and (75?1.5)% respectively; When MOI was 0.3125, 0.625, 1.25, the inhibition rate in CNHK600-p53 group and Ad-p53 group was (27?2. 5)% , (30?3. 7)% , (61?4. 3)% and(4?2.7)%, (5?3.5)%, (16?4.5)% respectively. Conclusion For hepatocarcinoma cell line PLC/PRF5 the effect of CNHK600-p53 is stronger than Ad-p53.
ABSTRACT
Objective To construct replication-deficient recombinant adenoviruses AdHNF4?that co-expresse human hepatocyte nuclear factor 4?(HNF4?) and green fluorescent protein(GFP) gene,and to evaluate the effect of HNF4?up-regulation on hepatocyte gene expression.Methods The HNF4?cDNA was obtained through RT-PCR from human hepatocyte.The recombinant adenoviral plasmid- pAdHNF4?was established using AdEasy system and packed in 293 cells.After transfection of human hepatoma cell lines HepG2 and Hep3B with AdHNF4?,the expression of HNF4?and other liver-associ- ated functional genes were evaluated by RT-PCR and Western blot.Results The recombinant plasmid pAdHNF4?was confirmed by restriction endonuclease digestion and sequencing.GFP expression was observed on the fourth day after packing the linearized pAdHNF4?in 293 cells.Stable transfection of AdHNF4?with a titer of 1?10~(10) efu/ml was obtained after repeated amplification.More than 90% of human hepatoma cells had GFP expression in 72 hours after transfection of AdHNF4?.The expression of HNF4?mRNA and protein were significantly up-regulated compared with the control group(3.4 folds in HepG2 infected with AdHNF4a and 5.2 folds in Hep3B infected with AdHNF4?).Furthermore,the transcriptional expressions of some liver-associated functional genes such as apolipoprotein,cytochrome P450 families,glutamine synthetase,phosphoenolpyruvate carboxykinase and glucose-6-phosphatase also increased after transfection of the virus,and the apoptosis ratio of the cells increased.Conclusions Up- regulating the expression of HNF4?in human hepatoma cells with AdHNF4?could enhance normal liver- specific function.Our study would provide a new idea for the researches on gene regulation of transplan- ted hepatocytes.