| [Background]In 1997, Folkman put forward firstly a new insight that the tumor can be treated with antiangiogenic therapy. An avascular tumor rarely grows to a size of larger than 2-3mm~3 and contains up to a few million cells. Angiogenesis of the tumor appears to be the fundamental requirement for tumor growth, invasion, and metastasis. The status of balance of angiogenic stimulators and suppressors determines the fortune of these in situ avascular and dormant microcancers. Several naturally occurring inhibitors of angiogenesis have been identified including angiostatin. Angiostatin, which has been shown to inhibit the endothelial cells proliferation and migration in vitro, is compound of the former four Kringels of plasminogen. In addition, it has been shown to be among the most potent inhibitors of tumor-induced angiogenesis in vivo. Systemic therapy with angiostatin protein has been shown to suppress tumor-induced angiogenesis and tumor growth . Notably , these studies required daily parenteral injections of large amounts of recombinant protein, illustrating a common difficulty in the pharmacologic use of therapeutic proteins. Gene therapy approaches may have advantageous properties as methods for delivery of systemic protein drugs.More challenges have recently emerged against anti-angiogenic therapy. The promising anti-angiogenic therapy is under pressure and suspicion due to recent disappointing clinical results. In the present study, we put forward a new insight thattumor hypoxia is also hindrance for anti-angiogenic therapy, and blocking tumor hypoxia pathway by antisense Hypoxia-inducible-factor-la could render angiogenic therapy a strong helper to eradicate established tumors. [Objective]1. To observe the effects of angiostatin gene on implanted human primary hepatic carcinome of nude mouse.2. To observe the effects of angiostatin gene combined with antisense HIF-la gene on implanted human primary hepatic carcinome of nude mouse.[Materials and Methods]Forty-eight nude mice, 4-5 weeks old, were used. The cell line of human primary hepatic carcinoma , namely SMMC-7721 , was cultured at 37 °C in 1640 medium(GIBCO), supplemented with 10% calf serum. Tumors were established by injiection of 3 X 106 tumor cells into the back of nude mice, and growth determined by measuring the long and short diameters.The mice were classified randomly into four groups, injected respectively with empty plamid PcDNA3-. angiostatin plasmid, antisense HIF-la plasmid, angiostatin plasmid+antisense HIF-la plasmid. Once the tumors reached 0.4cm in diameter, they were injected with lOOul gene transfer solution(100ug expression plasmid). The transduction was mediated by cationic liposome DOTAP. For combinational treatment, reagents were delivered in a timed fashion, where angiostatin plasmid was injected first, followed by antisense HIF-la plasmid 24h later.Half of the nude mice were used to observe the tumor growth curve, the other half to obtain the tumor samples. The tumor samples were prepared in the 4th day after gene transfer to be used in the examinations of the expression of angiostatin > HIF-1 a and VEGF with immunohistochemistry and western blot analysis, of MVD in the tumor with immunohistochemistry, and of cell apoptosis with TUNEL staining.Results were expressed as mean values ± standard deviation (X±s). SNK test and x2 test were used for evaluating statistical significance, where a value less than 0.05 (P<0.05) denote statistical significance. The software SPSS 11.5 was used in...
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