| Objective:Gene therapy has become more and more popular in the treatment of hepatocellular carcinoma. We will construct the recombinant adenovirus vector with hTERT-HSV-TK and observe the lethal effect of Ad-hTERTp-HSV-TK/GCV system and the bystander effect on the hepatocellular carcinoma cells which is called HepG2.Methods:1.A recombinant replication defective adenoviral vector of Ad-hTERTp-HSV-TK was constructed via homologous recombination which both shuttle plasmid pSU-Tp-TK and adenovirus backbone plasmid pBHGE3 transfected into the HEK293 packaging cells. Then the Ad-hTERTp-HSV-TK was identified,amplified and purified through PCR.2.We detected the activity of the HepG2 cells and the L-02 cells through the methyl thiazolyl tetrazolium (MTT) and flowcytometery after they were transducted by the recombinant adenovirus , adding GCV of different concentrations.3. We observed the inhibition ratio on HepG2 cells after mixing the transducted HepG2 cells and the non-transducted HepG2 cells.Results:1.The recombinant replication defective adenoviral vector of Ad-hTERTp-HSV-TK were identified by PCR successfully. The viral titer was 1.5×1010pfu/ml after amplification and purification.2. The survival rate of the HepG2 cells was decreased following the increasement of the MOI and the concentration of GCV. The evidence shows that the decreasing survival rate of the HepG2 cells was followed by the increasing MOI and the adding concentration of GCV while the L-02 cells had no obvious change via MTT. The same result was confirmed by flowcytometery.3. The bystander effect was significant, the 90.44% of HepG2 cells was inhibited when 70% of HepG2 cells were transducted. Conclusion:1.A recombinant replication defective adenoviral vector of Ad-hTERTp-HSV-TK was constructed successfully.2.The Ad-hTERTp-HSV-TK system is targeted to inhibit the HepG2 cells significantly, but no influence on the L-02 cells.
|
PDF Full Text Request