The Expression Research Of Thymidine Phosphorylase/PD-ECGF In Tumor

Thymidine Phosphorylase (TP) (E.C. 2.4.2.4) catalyzed reaction that plays a role in maintaining the balance of the nucleotide pool is: Thymidine+phosphate<->Thymine+2-deoxy-D-ribose+ 1-phosphate;2-deoxy-D-ribose is produced in vivo by the catalytic action of thymidine phosphorylase (TP) on thymidine and has recently been identified as an endpthelial-cell chemo-attractant and angiogenesis inducing factor. TP, previously known only for its role in nucleotide salvage, is now known to be angiogenic. TP is highly expressed in tumors compared with most normal tissues and has been correlated with tumor growth, invasion and metastasis in clinical studies. The prodrugs of 5-FU such as Furtulon (5'-DFUR) and Capecitabine ( fluoropyrimidine) require activation by TP to give out the derivatives that inhibit thymidylate synthase, leading to cell death. TP-overexpressing tumor cells show a greater susceptibility to these chemotherapeutic agentsgreater susceptibility to these chemotherapeutic agents than do normal cells, which contain lower levels of the enzyme. The studies of TP in abroad focus on the high TP expression in tumor and on the mechanisms of TP-induced angiogenesis and the clinical research of anti-angiogenesis therapy targeting TP. But, there is less research that has been focused on the mechanisms of highly expressing of TP in tumor and the evaluation of application value of anti-angiogenesis therapy targeting TP.Research Objectives:1. Express and purify Thymidine phosphorylase by using bacterial expression vectors . Prepare, purify and detect anti-human Thymidine Phosphorylase polyclonal antibodies by immunizing rabbits and mice.2. Identification of TP expression in cells with different prolific potential. TP expression in carcinoma of gallbladder, bile duct carcinoma and normal gallbladder measured by RT-PCR and immune-blot. TP expression in the invading part and non-invading part of carcinoma of gastric carcinoma and pulmonary carcinoma were measured by RT-PCR and immunohistochemistry. TP expression in keloids and normal skins measured by RT-PCR and immunohistochemistry.3. Investigation of TP expression differences in vitro and in vivo, and study the influence of in vivo microenvironment to TP expression. TP expressionin keloids and normal skins, and that in the primary cultured fibroblasts from keloids and normal skins measured by RT-PCR, immune-blot and immunohistochemistry.4. Investigation of controlling mechanism of thymidine over the high TP expression in tumor. Replication of the effect in vitro by supplementing cell culture medium with exogenous thymidine to investigate the influence of thymidine to TP expression.5. Investigation of TP expression in the parts with low angiogenesis. TP expression in keloids, carcinoma of gallbladder and bile duct carcinoma measured by RT-PCR, immune-blot and immunohistochemistry.6. Investigate the secretion of TP in different cell-lines. Cultivating of different cell-lines in vitro and measuring of TP expression in supernatants.7. Construction of TP recombinant adenoviral Vector.Methods and results:1. Express and purify Thymidine phosphorylase by usingbacterial expression vectors. The Thymidine phosphorylase bacterial expression vectors have demonstrated high expression efficiency, and Thymidine phosphorylase was purified.2. Prepare anti-human Thymidine Phosphorylase polyclonal antibodies by immunizing rabbits and mice. Purify antibodies by protein-A affinity column. Detect the titre of antibodies by ELISA and value the antibodies by immune-blot. The titer of rabbit andmice anti-human Thymidine Phosphorylase antibodies were 1:10,000 and 1: 32,000 respectively.3. The investigation of Thymidine Phosphorylase expression in multi-tissue and cell-lines by RT-PCR, immune-blot and immunohistochemistry. The expression of TP is higher in keloids than in normal skins. The expression of TP is higher in tumors than norma...