| Bladder cancer is the most common urological malignant disease in China. How to prevent its recurrence after operation, control the residue and the in situ carcinoma is an urgent problem. Most research reported that angiogenesis is necessary for its nutrition supply and waste drainage till its volume reaches 3mm3 and the quantity of cell is 107. Otherwise, the tumor will get regressed. So it is a very promising way to inhibit tumor angiogenesis in order to control its growth and metastasis.Vascular formation is regulated by stimulating factors and inhibiting factors, and many stimulators and inhibitors have been identified by now. The most important stimulators are Vascular Endothelial Growth Factor (VEGF) and basic Fibroblast Growth Factor (bFGF), and inhibitors are endostatin, angiostatin, interferon and so on. Endostatin is a 20-kDa C-terminal proteolytic fragment of collagen X VEI. Among all the inhibitors, endostatin has been testified as the strongest one. It could inhibit the proliferation of endothelial cells, induce apoptosis of tumor cells, inhibit tumor growth and metastasis effectively, and has no side effect. So endostatin maybe the most preferred agent for tumor anti-angiogenic therapy.Endostatin has been applied for tumor biological treatment in two types: protein and gene. Proteinic endostatin has been verified for it's strong anti-angiogenesis function, but there are still some potential problems: 1. The difficulty of producing sufficiently large amounts of biologically active protein and keep its activity; 2. As a protein drug, endostatin tends to have a short half-life in vivo, thus posing concerns about maintaining therapeutically effective serumlevels; 3. the practice of transferring endostatin protein into patients body constantly is very difficult. Alternatively, gene therapy, which can deliver gene cassette encoding endostatin into mammalian cells, can make the cells express and secret endostatin successively, may has a good curative effect.Gene therapy with endostatin has been tried with several vectors: plasmid for muscular injection, liposome transfection and virus vectors mediated gene delivery, all the methods are valid for some extent. Adeno-associated virus(AAV), a kind of Parvoviridae and naturally replication defective virus, is the only mammalian virus which could integrate into the No. 19 chromosome point specifically, and could avoid the latent risk of host cell mutation caused by random integration. AAV vector has many advantages in tumor anti-angiogenic gene therapy, and has become a hot spot these years.The infiltrative growth and metastasis of bladder cancer are angiogenesis dependant like other solid tumors, somebody even considered Micro-Vessel density(MVD) is an independent prognosis standard. We adopt AAV-2 virus as a gene therapy vector to mediated IgG leader sequence and endostatin cDNA, the interesting sequence will be integrate into the No. 19 chromosome point specifically after the virus infected bladder cancer cells, then the cells will express endostatin constantly and secret it into the extracelluar matrix.Part 1 The package of rAAV-EndostatinConstructing AAV Vector plasmids with gene engineering and molecular biological techniques, packaging rAAV-Endostatin with plasmids co-transfection techniques:1. The interesting sequence was derived from polymerase chain reaction(PCR) amplification of plasmid pIRES-Endo which including human IgG-Endostatin using a pair of primers for the mature form of the cytokine. The sequence of the sense primer was GACatcgatATGAAATGCAGC TGGGTTATC (ClaI site in lowercases), and the antisense primer was TATggatccCTA CTTGGAGGCAGTCATG(BamHI site in lowercases). The cDNA segment was verified by sequencing, then it was double digested by BamHI and ClaI, the product was reclaimed. The plasmid pCMV-MCS was also double digested by the same enzymes and ligated with IgG-Endostatin cDNA to construct plasmid pCMV-IgG-Endostatin. DH5 a E.coli was transformed with the constructed plasmid pCMV-IgG-Endostatin according to the manufac... |
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