The Construction Of Double-suicide Gene Vector Driven By PSMA Promoter And Its Expression In Vitro And In Vivo

Gene-directed enzyme prodrug therapy (GDEPT) is one of the noticeable new progresses in the research of antibody-directed treatment for tumor in recent years.The prodrug, a kind of substances has no or only lower, can be converted into actived drug when catalyzed in vivo. The virus vector or non- virus vector which carry the the specific activating enzyme to catalyze the prodrug ,can selectively conjugate to the target maligant tissue at the same time. Then the prodrug can be activated by the enzyme, which is carried by the virus vector or non- virus vector, and converted into actived cytotoxitic molecules located at a certain region in the tumor tissue to play the cytotoxitic effect on the maligant cells and kill them only ultimately.Prostate cancer , one of the most prevalent maligant tumors in older males, takes the first place for American old man on the list of male tumor incidence rate. The incidence rate has been increasing year by year with change of average life span and dietary structure in China.For gene therapy on prostate cancer cells, it is essential touse a strong and tissue-specific promoter for suicide genes to be selectively expressed on the cancer cells. Furthermore, in the case of prostate cancer, it is important to note that most patients have already received androgen ablation therapy. Prostate-specific antigen (PSA) has a strong promoter activity, however the expression of PSA is strictly regulated by androgen, and the PSA promoter activity is greatly reduced under androgen ablation conditions. The expression of PSMA is independent of androgen. Previous observation have demonstrated that the level of PSMA expression in prostate cancer was upregulated after androgen ablation therapy. As a result, the PSMA promoter apperas to be highly suitable for gene therapy.Suicide gene transfer is used as one of the most promising strategies for the therapy of prostate cancer. The most widely used genes include thymidine kinase (TK) and cytosine deaminase ( CD). The suicide gene system is safer than other strategies to kill cells because it can be easily regulated by this "two step" treatment.PSMA promoter was amplified by polymerase chain reaction (PCR) , The pCMV promoter on eukaryotic expression vector pIRES was replaced by PSMA promoter and PSMAP-IRES vector was obtained. The recombinant plasimd PSMAP-IRES was identified by restriction analysis and DNA sequence. Sequence and restriction analysis showed that PSMA promoter by amplified was identical with that of GenBank reported(GenBank accession number AF007544). Then the gene FCY1 and TK was cloned into corresponding sites of PSMA-IRES and the...