Research On Oncolytic Adenovirus Mediated Gene Therapy Of Hepatocellular Carcinoma And Metastatic Tumor In Vitro And In Vivo

Objective: To construct a recombinant oncolytic adenovirus and research its effect and mechanism on gene therapy of hepatocellular carcinomaMethods: To design and synthesize one pair of primers according to declared Ad5 E1A gene sequence from GenBank. The E1A gene fragment was obtained by PCR with QBI-293A cell genome DNA as template. It was cloned into pUCm-T vector and sequenced. After enzyme digestion, the E1A gene was ligated to transfer vector pAdTrack-CMV. The positive clone pAdTrack-CMV-E1A were lineared by PmeI and co-transformed with pAdeasy-1in BJ5183 E.coli. The recombinant adenovirus vector pAdeasy-1-pAdTrack-CMV-E1A were digested by PacI and transfected into QBI-293A cells with liposomes. The oncolytic recombinant adenovirus rAd-E1A was obtained. It infected HepG2 and SMMC-7721 human hepatocellular carcinoma (HCC) cells and normal liver cell line HL-7702. The oncolytic effect was observed under microscope and the growth inhibition and apoptosis-inducing were detected by MTT and FCAS. The mechanism of rAd-E1A was identified by RT-PCR and Western-blotting. The effect and mechanism of rAd-E1A on gene therapy of hepatocellular carcinoma in vivo was observed through the SMMC-7721 human liver cancer subcutaneous model in nu/nu mice. The effect of rAd-E1A on antitumor inhibitive ratio, thymus index, spleen index, white blood cell, lymphocyte, monocyte and neutrophil were detected in mouse ascitic hepatoma subcutaneous model. It infected the ECV304 and the cell growth, secretion of VEGF and expression of NF-κB were detected by MTT, ELISA and Western-blotting respectively.Results: Full encoding sequence of E1A and its ORF identified with that reported in GenBank was obtained successfully.The recombined oncolytic adenovirus rAd-E1A was obtained. It can replicate,...