| Adult endothelium is usually quiescent, but in response to physiological or pathological stimuli (such as injury, tumour growth, or diabetic retinopathy), the endothelium can alter to a proliferating status. Tumor angiogenesis is critical for the tumor growth and matastasis. Primary cancers can not grow above 1-2 mm in diameter until its own neovasculization is formed. Tumor angiogenesis involves complex interactions between tumoral, stromal, endothelial cells, fibroblasts and the extracellular matrix. Angiogenesis depends on the balance of proangiogenic and antiangiogenic factors. Tumor angiogenesis is regulated by a balance of stimulators (e.g., VEGF, bFGF) and inhibitors of angiogenesis (e.g.angiostatin, endostatin). It is called "angiogenesis switch" . Anti-angiogenesisis a reliable therapeutic approach for cancer treatment. Endostatin is a specific inhibitor of endothelial cell growth. Endostatin couldhave both local and systemic effects and possibly protect against metastatic dissemination in vivo. Endostatin has been used successfully in vitro for it has a series of advantages such as broad-spectrum action, direct endothelial targeting, low toxicity, absence of drug resistence after repeatly used. To promote tumor regression and to maintain dormancy, antiangiogenic agents need to be chronically administered. The difficulties in protein production, long-term storage of bioactive protein, and the cumbersome daily administration may be overcome through transfer of the endostatin gene. Therefore, continuous overexpression of antiangiogenic factors by gene therapy, for instance, should counteract the tumor-induced angiogenesis. Gene therapy offers a potential way to achieve sustained therapeutic release of potent antiangiogenic substances. As a step toward this goal, we have generated recombinant adenovirus vectors (rAd-ES) that carry genes coding for endostatin. Several biologic features of adenovirus have made such viruses the vectors of choice for certain of these applications. For example, adenoviruses transfer genes to a broad spectrum of cell types, and gene transfer is not dependent on active cell division. Additionally, high titers of viruses and high levels of transgene expression generally can be obtained.Angiogenesis is a bad prognostic factor of bladder cancer. Treatment targeting neovasculization is a good choice. This study is designed for the basic rescerch of the inhibition of endostatin for bladder cancer, and look forward the clinical application.The first part of this study: the constrction of adenovirus shuttle vector coding endostatin. First, design specific primer based on the sequence of endostatin (ES) in the Genbank, amplify the DNA fragment(within the restriction endonuclease sites as same as the shuttle vector pAdTrack-CMV). The pAdTrackCMV-ES was made from pAdTrack-CMV and ES by cut and ligation. A recombinant adenoviral plasmid (prAd-ES) is generated from pAdTrackCMV-ES and adenovirus backbone plasmid called pAdEasy-1 using homologous recombination in bacteria BJ5183. And then prAd-ES is amplified in DH10B cells.The second part of this study: the constrction of the recombinant adenovirus. Mediated by liposome, the recombinant adenovirus plsmid prAd-ES transfect the 293 cells (human embrotic kidney cells, containing the El region that is essencial for the virus replication). In the 293 cells, the virus packaged and amplified itself.We can trace this course by observe the GFP(green fluorescent protein) through the fluorescent microscope.The recombinant virus was purified byCsCl gradient centrifuge and tittered by TCID50 method.The third part of this study: the inhibit effect of endostatin on tumor in vivo and in vitro. The cellular culture supernatant of rAd-ES has significant inhibit effect on umbilical vessel endothelial cells ECV-304. It has significantly statistic difference between rAd-ES and its control-AdLacZ. We constrct the nude mice(SCID) model through the subcutaneously implanting the EJ cells of the human bladder. Injecting the cellular culture sup... |
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