岩藻糖基化海参硫酸软骨素结构的质谱分析和抗肿瘤活性研究

项目名称： 岩藻糖基化海参硫酸软骨素结构的质谱分析和抗肿瘤活性研究

项目编号： No.30871944

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 电工技术

项目作者： 薛长湖

作者单位： 中国海洋大学

项目金额： 30万元

中文摘要： 以美国肉参和菲律宾刺参为研究对象，采用现代生化分离技术，分离纯化岩藻糖基化海参硫酸软骨素（fucosylated chondroitin sulfate from sea cucumber, SC-CHS）；采用稀酸法、酶法和氧化降解法等手段对SC-CHS进行可控降解，获得富含硫酸岩藻糖支链的SC-CHS寡糖混合物，通过生化分离技术对其分离、纯化；采用电喷雾质谱/质谱技术解析所得寡糖序列，并结合NMR、甲基化技术确定SC-CHS结构；利用体外培养肿瘤细胞株和建立肿瘤动物模型,研究SC-CHS寡糖的抗肿瘤活性，进而确定SC-CHS的构效关系。在此基础上，以肿瘤细胞乙酰肝素酶的基因和蛋白表达为切入点，阐明SC-CHS抗肿瘤的分子机制。通过本课题的研究，将建立质谱法分析SC-CHS寡糖的理论基础，填补国内外研究空白；对SC-CHS抑癌活性的深入研究，为开发新的海洋多糖药物提供了理论依据。

中文关键词： 海参；岩藻糖基化海参硫酸软骨素；电喷雾质谱/质谱 ；抗肿瘤

英文摘要： The fucosylated chondroitin sulfate of sea cucumber(SC-CHS), which has a chondroitin sulfate-like structure containing large numbers of sulfated fucose branches, was isolated and purified from the body wall of the sea cucumber. The topic of this research included that the sequences of the purified oligosaccharides were determined by tandom mass spectrometry, the structural determination of SC-CHS was further completed and confirmed by NMR, IR and methylation analysis. The anti-tumor effects of SC-CHS were investigated by the several cancer cell lines in vitro and the model of murine Lewis lung carcinoma in vivo. The significant results obtained after 3 years study included:sulfate substitutions pattern on sulfated fucose branches of SC-CHS varied from different sea cucumber species were ascertained by 1H NMR for the first time. On this basis, the fingerprint chromatography of polysaccharides from sea cucumber was established. The methods of preparation of oligosaccharides with rich sulfated fucose branches from SC-CHS using peroxide oxidation and sequences analysis of the oligosaccharides using ESI-MS-MS were established for the first time. The structures of these highly sulfated oligosaccharides were identified by using ESI-MS/MS combined with NMR. The structure of SC-CHS from sea cucumber Isostichopus badionotus were deduced to be: -[3GalNAcβ,6S)1-4GlcAβFucα,4S)1-3) 1]n -. The anti-tumor metastasis effect of SC-CHS was demonstrated and the molecular mechanism was also elucidated. The anticoagulant activities of SC-CHS and oligosaccharides with sulfated fucose branches from SC-CHS were evaluate. The anticoagulant pathway, structure-activity relationship and minimum unit of ligosaccharides were clarified. These results of this research would provide academic support for the exploitation of SC-CHS and its oligosaccharides. Also, the results would fill the gap in the research of sulfate polysaccharides from sea cucumber.

英文关键词： sea cucumber;The fucosylated chondroitin sulfate of sea cucumber;ESI-MS-MS;anti-tumor activity