鲨鱼软骨抗血管生成多肽的分离纯化及其生物活性

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	鲨鱼软骨抗血管生成多肽的分离纯化及其生物活性
其他题名	Polypeptide from Shark Cartilage with Potent Anti-angiogenic Activity
	郑兰红
学位类型	博士
	2007-06-15
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	鲨鱼软骨多肽新生血管生成分离纯化 N-末端序列
摘要	以血管生成为靶点的抗肿瘤策略是抗肿瘤领域的研究热点，目前已经发现许多天然和化学合成的抗血管生成药物。鲨鱼软骨作为抗新生血管生成因子的重要来源的研究已有20多年的历史，很多研究显示鲨鱼软骨提取物有抗血管生成活性。但鲨鱼软骨活性多肽的完整分子结构一直未见报道；鲨鱼软骨活性多肽干扰血管生成通路的信号途径尚不明确。本文应用盐酸胍抽提、丙酮分级沉淀、超滤、凝胶层析等分离技术，从青鲨（Prionace glauca）软骨中分离纯化并鉴定了一种新的具有抗新生血管生成活性的多肽。经SDS-PAGE和N-末端氨基酸序列分析显示，该多肽分子量为15500 Da，采用蛋白数据库分析表明该多肽是一种新发现的鲨鱼软骨多肽（Polypeptide from Prionace glauca，PG155）。体外实验显示，PG155抑制内皮细胞生长因子（vascular endothelial growth factor，VEGF）介导的人脐静脉内皮细胞（human umbilical vein endothelial cell ，HUVEC）迁移和管腔形成，并呈剂量依赖关系。200 μg/ml PG155对牛主动脉内皮细胞（Bovine Aortic Endothelial Cells，BAECs）和HUVECs及以下癌细胞，包括人肝癌细胞（human hepatoma Bel-7402 cells，Bel-7402）、口腔上皮癌细胞（human oral epidermoid carcinoma KB cells ，KB）、人结肠癌细胞（human colon cancer HCT-18 cells，HCT-18）和人乳腺癌细胞（human breast MCF7 cancer cells ，MCF7）的增殖均无抑制作用，说明PG155无细胞毒作用。20 μg/ml PG155显著抑制HUVEC的迁移和管腔形成；40-80 μg/ml PG155 对VEGF 介导的HUVEC的迁移和管腔形成几乎完全抑制。体内实验显示，PG155显著抑制斑马鱼胚胎模型新生血管生成，并呈剂量依赖关系。形态学观察表明PG155显著抑制斑马鱼胚胎肠下静脉（subintestinal vessels， SIVs）的生长，随着浓度的升高SIVs的生长可受到完全抑制。碱性磷酸酶染色分析显示，在一定浓度范围内，PG155随着浓度的升高对斑马鱼胚胎整体血管生成抑制作用依次增强。160 μg/ml PG155会引起斑马鱼胚胎心脏功能障碍。由海洋生物中发现新的肿瘤新生血管生成抑制剂国内外的报道较少，我们的工作表明鲨鱼软骨可作为血管生成抑制剂的重要来源，鲨鱼软骨活性多肽PG155由于具有极低的细胞毒作用，并能抑制VEGF介导的血管生成过程，有希望成为一类新型抗肿瘤药物。
其他摘要	Anti-angiogenesis as a promising anticancer strategy has led to the discovery of many natural and synthetic anticancer agents. Shark cartilage has been investigated as a source of anti-angiogenesis agents for more than 20 years. Numerous studies have confirmed that shark cartilage extract possesses anti-angiogenic activity. However, little is known about the exact composition of polypeptides. And also, there is very little information about the mode of action on anti-angiogenesis of polypeptides from shark cartilage. Using guanidine-HCl extraction, acetone precipitation, ultra-filtration and chromatography, a novel polypeptide with potent anti-angiogenic activity was purified from cartilage of the shark, Prionace glauca. N-terminal amino acid sequence analysis and SDS-PAGE revealed that the substance is a novel shark cartilage polypeptide with MW 15500 (designated name, PG155). Polypeptide PG155 inhibited vascular endothelial growth factor (VEGF) induced tube formation and migration of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner in vitro. Treatment with 200 μg/ml PG155 did not affect the growth of Bovine Aortic Endothelial Cells (BAECs) as well as HUVECs. Also we tested if PG155 inhibited the proliferation of cancer cells, including human hepatoma Bel-7402 cells (Bel-7402), human oral epidermoid carcinoma KB cells (KB), human colon cancer HCT-18 cells (HCT-18), as well as human breast MCF7 cancer cells (MCF7), and no cytotoxic effects was found. Transwell experiment revealed that the polypeptide PG155 inhibited VEGF-induced migration of HUVECs. Exposure of HUVECs in 20 μg/ml PG155 significantly decreased the density of migrated cells and the tube formation of HUVECs. Almost complete inhibition of cell migration and tube formation was found when HUVECs were treated with 40-80 μg/ml PG155. Treatment of the zebrafish embryos with PG155 resulted in a significant reduction in vessel formation when introduced to zebrafish embryos prior to the onset of angiogenesis. Morphologic observation showed that PG155 markedly inhibited the growth of subintestinal vessels (SIVs) of zebrafish embroy. A higher dose resulted in almost complete inhibition of SIVs growth, as observed by endogenous alkaline phosphatase (EAP) staining assay. A dose-dependent effect was also found when treatment of zebrafish embroys with PG155 at different concentrations. 160 μg/ml PG155 inhibited heart development of zebafish embryo. There is little information available concerning the new tumor angiogenesis inhibitors (TAIs) extracted from marine organism. Our observations indicated that shark cartilage may be as an important source of TAI. The new polypeptide, PG155, as a specific angiogenic inhibitor, may be developed as a novel kind of anti-tumor agent.
页数	105
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1323
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	郑兰红. 鲨鱼软骨抗血管生成多肽的分离纯化及其生物活性[D]. 海洋研究所. 中国科学院海洋研究所,2007.

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