【作者】 王秀红；

【导师】 李玉新； 乌垠；

【作者基本信息】 东北师范大学 ， 遗传学， 2005， 硕士

【摘要】 血管生成是指从已存在的血管网中长出新血管的过程。它是一个复杂多步骤的过程,包括血管内皮的增殖、迁移、分化,胞外基质降解,管状形成,新生毛细血管分支生芽等。许多实验已经表明肿瘤的生长、浸润和转移等均依赖新生血管的形成。体内血管生成过程是血管生成刺激因子和血管生成抑制因子共同调控的,血管生成趋向的形成是肿瘤发展过程中的关键事件。因此,抗血管生成疗法在治疗肿瘤中是非常有前途的。抑制血管生成不仅可以抑制肿瘤生长,还可以使肿瘤衰退到“非血管生长型”时的体积。现已发现了多种以肿瘤血管内皮细胞为靶向的抗血管生成药物,血管抑素(Angiostatin)就是最早发现的血管生成抑制因子之一。它是纤溶酶原分子酶解产生的内部片段,包括纤溶酶原 5 个 Kringle 结构中的前三个或前四个。血管抑素最初被发现是由于它在荷瘤小鼠中存在,并能够抑制人和动物的多种肿瘤的生长和转移。之后发现纤溶酶原分子中几乎每个 Kringle 结构在体内、体外实验中都具有一定的抗血管生成功能,尽管纤溶酶原本身并没有这种功能。通过基因重组或纤溶酶原分子酶解产生的各个 Kringle 结构及多种 Kringle 组合已被广泛用于抗血管生成研究。血管抑素可显著抑制体外培养的血管内皮细胞增殖和迁移,诱导内皮细胞凋亡。多种研究表明,其在动物模型中可诱导肿瘤血管衰退,能够完全抑制多种原发瘤和转移瘤的生长。血管抑素治疗肿瘤是非常有潜力的,目前处于临床实验阶段。 我们已经通过基因重组技术大量表达出重组人血管抑素 K1-3(rhK1-3)。为研制开发 rhK1-3,本实验通过建立体内、体外血管生成模型来检测 rhK1-3 的生物学活性。用原代培养的人脐静脉内皮(HUVEC)建立体外实验模型,采用 MTT 法体外检测 rhK1-3 的生物学活性。实验证明了 rhK1-3 对 HUVEC 的生长活性有较强的抑制作用,并存在一定的剂量及时效关系。研究了 rhK1-3 对 bFGF 刺激的 HUVEC 增殖活性的影响,结果表明rhK1-3 可对抗 bFGF 的作用。此外,研究了纤溶酶原对体外培养的 HUVEC 的作用。结果表明,纤溶酶原对体外 HUVEC 的生长活性没有影响,同时也不影响 rhK1-3 对 HUVEC 的抑制作用。 创造性地建立了鸡胚卵黄囊膜(yolk sac membrane, YSM)血管生成模型,在器官水平上研究了 rhK1-3 对鸡胚 YSM 血管生成的影响。实验采取了两种不同的培养方法(开窗法和玻璃杯培养法)和两种不同的给药方式(混合纤维素微孔滤膜给药法和直接给药法)定性观察了 rhK1-3 对鸡胚 YSM 血管生成的影响。比较了两种不同的培养方法的优缺点,同时探讨了两种不同给药方式的优缺点。实验采用玻璃杯培养法培养鸡胚,混合纤维素微孔滤膜作为药物载体建立鸡胚 YSM 血管生成定量模型,采用专门设计的计算机图形分析软件对 rhK1-3 的生物学活性进行了定量研究。采用了多个血管生成定量指标,能够较客观、真实地反映 rhK1-3 对鸡胚 YSM 血管生成的影响。实验表明,rhK1-3 对 YSM血管生成有较强的抑制作用,表现为给药区血管密度明显降低,给药的鸡胚发育迟缓等。III鸡胚 YSM 血管生成模型的建立为与血管生成相关药物的筛选提供了一简便、快速、准确的体内实验模型。本实验为重组人血管抑素 rhK1-3 研制开发和临床应用提供了实验依据。rhK1-3 具有较高的抑制血管生成活性,是理想的抗肿瘤药物。一旦投入应用,将使肿瘤治疗水平上升到一个新的高度。关键词:重组人血管抑素 rhK1-3;血管生成;Kringle 结构;卵黄囊膜;绒毛尿囊膜更多还原

【Abstract】 Angiogenesis, the outgrowth of new capillaries from pre-existing vessels, is a complexmultistep process that includes endothelial cell proliferation, migration and differention,degradation of extracellular matrices, tube formation, and sprouting of new capillary branches.Several recent studies have shown that tumor growth, local invision, and metastaticdissemination are all dependent on the formation of new microvessels. The process ofangiogenesis is regulated by a balance between pro-angiogenic and anti-angiogenic factors,and the shift to an angiogenic phenotype is a key event in tumor progression. As a result,antiangiogenic therapy is a highly promising new strategy in the treatment of tumor.Inhibition of angiogenesis not only arrests tumor growth, but also results in tumor shrinkageto a prevascular size. Many antiangiogenic agents have been developed that are directedagainst the endothelial cells of the tumor blood vessels. One of the first describedangiogenesis inhibitors was angiostatin, a internal proteolytical fragment of plasminogen,consisting of the first three or first four Kringle domains of plasmiongen. Angiostatin wasinitially discovered by its ability to prevent the growth of both human and murine tumormetastases in mice. Since then it has been shown that each Kringle domain of plasminogenmolecular has some antiangiogenic activity in vitro and in vivo, although plasminogen itselfhas not the activity. The antiangiogenic functions of Kringles of plasmiongen have beenextensively studies using recombinant plasminogen Kringles and Kringle fragments fromproteolytical processing of native plasminogen. Angiostatin has been reported to effectivelysuppress the endothelial cell growth, induce the endothelial cell apoptosis and blocks themigration of endothelial cell in vitro. Many researches have demonstrated that angiostatin caninduce tumor vasculature regression, which leads to a complete cessation of a variety ofprimary and metastatic tumor growth in animal models. Angiostatin has promising therapeuticpotential and is now in clinical trials. We have already obtained a large quantity of recombinant human angiostatin K1-3(rhK1-3). The protein expressed in the form of inclusion body must be renatured to gain itsbiological activity. In order to develop the gene engineering durg, it was necessary to study itsin vitro and in vivo biological activity. In this study, we used primarily cultured humanumbilical vein endothelial cell (HUVEC) to detect the inhibition activity of rhK1-3. Inhibitionof cell growth was observed by MTT colorimetric assay. It was found that rhK1-3 stronglyinhibited the growth of HUVEC in a dose-time-dependent manner in vitro. We also found thatrhK1-3 can inhibit the growth activity of HUVEC promoted by bFGF, which showed thatrhK1-3 can antagonize the action of bFGF. Plasminogen was added to the cluture medium tostudy its effect on HUVEC and the activity of rhK1-3. Plasminogen appeared to have noeffect on them in vitro. We creatively used yolk sac membrane (YSM) to assess the antiangiogenic activity ofrhK1-3 in vivo. Two different methods (making a hole on the shell and putting the chickembryo in the beaker) were used to incubate chick embryo. We adopted two ways (takingmixed cellulose micropore filter membrane as drug carrier and directly adding drug to thesurface of YSM) to add the rhK1-3 to the YSM blood vessels. It was found that rhK1-3caused significant decrease in vessel density and caused microvascular corrosion. In addition,rhK1-3 delayed the chick embryo development. A quantitative model of angiogenesis hasbeen established using beaker to incubate the chick embryo and adopting micropore filtermembrane as drug carrier. The YSM model is convenient to quantify the angiogenesis ofchick embryo YSM. The use of specially designed image analyzer and adoption several indexto analyze the antiangiogenic activity made the result more objective and more reliable. TheYSM model we established in this study is more convenient for in vivo qualitative evaluationsof angiogenesis than CAM model which is更多还原