论文标题:Angiostatin基因联合反义HIF-1α基因治疗人肝癌裸鼠移植瘤的实验研究
Experimental Study of Treating Impanted Human Primary Hepatic Carcinoma of Nude Mice by Angiostatin Gene and Antisense HIF-1α Gene In-Vivo
论文作者 董典宁
论文导师 寿楠海,论文学位 博士,论文专业 外科学
论文单位 山东大学,点击次数 99,论文页数 65页File Size10723k
2004-04-12论文网 http://www.lw23.com/lunwen_352418902/ Angiostatin HIF-1α;基因治疗;反义基因治疗;抗血管生成治疗;裸鼠;肝癌
Angiostatin; HIF-1α; Nude Mouse; Liver Cancer; Gene Therapy; Antisense Gene Therapy; Antiangiogenic therapy
[背景] 1971年Folkman首先提出用抑制肿瘤血管生成的方法治疗肿瘤。他认为实体瘤形成之初无血管生成,此时肿瘤细胞的营养主要通过弥散获得,当细胞距离毛细血管200μm以上时,氧气将无法通过弥散作用抵达肿瘤细胞,因此当肿瘤的细胞数达到1×10~7个以后,弥散获取的营养已无法满足肿瘤存活和生长的需要,此时如再无新生血管的生成,肿瘤组织将发生退化。而若有新生血管长入肿瘤组织,肿瘤将迅速生长,并且其浸润和转移能力也大大加强,因此血管生成是肿瘤发展、转移的重要条件,也是所有恶性肿瘤的共性。阻断肿瘤新生血管形成就可能阻止肿瘤生长和转移,因而抗肿瘤新生血管疗法成为了肿瘤治疗新的研究热点。 近期人们对此策略投入了越来越多的希望,发现了多种血管生成抑制因子,其中Angiostatin(血管抑素)更是引人注目。它由纤溶酶原(plasminogen)前4个Kringles(简称Ks)片段组成,具有显著抑制血管内皮细胞增殖的活性。荷瘤小鼠体内注射该蛋白能抑制其皮下多种人源性或鼠源性实体瘤生长。尽管血管抑素有很强的抗肿瘤作用,但是要达到治疗效果,需大剂量、长期持续应用,周期长、花费大,并且存在一定程度的副作用。因此,抗血管生成的基因治疗逐渐受到人们的重视。它克服了单纯注射蛋白质的缺点,使得各种血管生成抑制因子能够在组织中持续稳定的表达。尽管在具体应用过程中存在着很多问题,但是目前的多项研究结果表明这是一种充满生机活力的肿瘤治疗手段之一。
[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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