Construction Of A Solid Tumor Anerobic Targeting And Gene-directed Enzyme Prodrug Anticancer Therapy System And The Experimental Study For Its Anticancer Effects

Gene-directed enzyme prodrug therapy(GDEPT) is a highlight of the tumor gene therapy nowadays, and many progresses have been achieved in this field. This gene therapy method mostly use virus as the gene vectors, so far the virus are still the most effective gene vector, but the virus-mediated gene transferring in GDEPT isn't very efficient and the tumor-targeting is also relatively poor. So the tumor gene therapy is badly impeded by the deficiency of a fine gene vector. The center of solid tumor is generally at low level of oxygen, and anaerobic bacteria have a tendency to colonize in low oxygen environment, so the anaerobic bacteria can possibly be used as a target vector for tumor gene therapy. Bifidobacterium Infantis has been proved to be good targeting toward mice melanoma in this laboratory andnon-pathogenic, therefore it was chosen as a gene transferring vector in this study. Anti-tumor prodrug convertase gene, the cytosine deaminase (CD) gene, was integrated into the bacteria genome, so the gene transferring vector system carrying recombined CD was constructed. In vitro study, this system with anti-tumor prodrug 5-FC showed good killing effect on mice melanoma B16-F10 cells, and the animal experiment on mice melanoma model was also showed significant tumor-inhibitive effect. The study showed that 1) the recombined plasmid with CD and pGEX-1LambdaT fragments was reconstructed, and transferred into Bifidobacterium Infantis, the extracts from positive colony was enzymed and electrophoreses, two bands, which were similar with CD and pGEX-1LambdaT fragments respectively, were harvested. 2) PCR and RT-PCR identification showed that foreign CD gene had been integrated into genome of positive colony, and its mRNA expression was tested positive. 3) In vitro, tumor cells in test groups appeared remarkable damaging changes on morphology, and the cell growth was significantly inhibited, while the control group didn't . 4) Experiment on mice melanoma model indicated that treated with this recombined bacteria and 5-FC in three weeks observing period, the tumor growth span was remarkably prolonged and the tumor mass was also significantly inhibited compared with control group, The differences were more and more obvious along with the times by.The result of this study indicated that the solid tumor targeting gene transferring system, which was mediated by Bifidobacterium Infantis carrying CD gene, was successfully constructed. Both in vivo and vitro, this system could express cytosine deaminase which was responsible for converting 5-FC to 5-FU. Treated with 5-FC, this system displayed a significant inhibitive effect on tumor in vivo and vitro. This gene therapy system, Bifidobacterium Infantis mediated gene directed enzyme prodrug anticancer therapy system, may have a valuable prosperity in the field oftumor targeting gene therapy.