Research On Anti-tumor Effect In Vivo And In Vitro Of ING4 And PTEN Adenovirus

Objective: To construct a recombinant ING4 and PTEN adenovirus and research its effect and mechanism on gene therapy of lung carcinoma.Methods: On the basis of pcDNA3.0-mING4 and hING4 amino acid sequence, mING4 gene was reconstitution by site-specific mutagenesis technique,.After enzyme digestion, the ING4 gene was ligated to transfer vector pAdTrack-CMV. The positive clone pAdTrack-CMV-ING4 were lineared by PmeI and co-transformed with pAdeasy-1in BJ5183 E.coli. The recombinant adenovirus vector pAdeasy-1-pAdTrack- CMV-ING4 were digested by PacI and transfected into QBI-293A cells with liposomes. The recombinant adenovirus Ad-ING4 was obtained, and by the same way, the recombinant adenovirus Ad-PTEN was obtained. The Ad-ING4 and Ad-PTEN infected A549 lung carcinoma cells. The effect was observed under microscope and the growth inhibition and apoptosis-inducing were detected by MTT and FCAS. The Metastasis and infiltration ability of A549 cells was observed by using Transwell Chanber. The mechanism of Ad-ING4 and Ad-PTEN was identified by RT-PCR and Western-blotting and ELISA. The effect and mechanism of Ad-ING4 and Ad-PTEN on gene therapy of lung carcinoma in vivo was observed through the A549 human lung cancer subcutaneous model in nu/nu mice. The Ad-ING4 and Ad-PTEN infected the ECV304 and the cell growth, secretion of VEGF were detected by MTT,and ELISA respectively.Results: Humanize reconstitution ING4 gene and PTEN identified with that reported in GenBank was obtained successfully.The recombined adenovirus Ad-ING4 and Ad-PTEN was obtained. They can induce apoptosis and inhibit growth in A549cells in vitro and in vivo, and Ad-ING4 influence the metastasis and infiltration ability of A549 cells obviously. Ad-ING4 can up-regulation apoptosis-associated P21, but not...