深海真菌来源呋喃半乳寡糖的结构鉴定及其激活巨噬细胞的分子机制研究

In addition to special structure, the characteristic marine oligosaccharides have a wide range of bioactivity and have very wide development and application prospects in medicine. Exploitation and utilization specific and unique marine oligosaccharides from the various marine organisms is still an important research direction of marine drugs. . A novel galactomannan was isolated in our laboratory from the broth of a deep-sea fungus, which contained a long chain galactose oligosaccharides linked to a mannan core. Different with the normal galactose in the higher organisms, the galactose of this fungus exopolysaccharide had the furan configuration. Recently, the research on the structure and function of galactofuranose oligosaccharides is rarely reported. Our early research had shown that the mixture of the galactofuranose oligosaccharides is of significant biological activity in activating mice macrophages. For further research on the structural characteristics and functional properties of galactofuranose oligosaccharides, it is considered to obtain galactofuranose oligosaccharides with different degrees of polymerization through controlled partial acid hydrolysis method, analyze the structure of the oligosaccharides at the support of MS and GC-MS and NMR technology. On this basis, through measuring the breaking rules of the secondary ion mass spectrometry, an efficient and rapid method for trace analysis of this kind of oligosaccharides by ESI-MS/MS is to be established. After preparation of galactofuranose oligosaccharides of different degrees of polymerization by HPGPC, the relation between the immunoregulation activity and the degree of polymerization will be measured to get the best active fragment. Then the regulation mechanism on macrophages will be furtherly explored including the recognition between the oligosaccharide and the cell surface receptor and the effect on the related cell factor.. The research project will systematically study the structure and biological activity of galactofuranose oligosaccharides for the first time, which not only enrich marine oligosaccharides resources and expand the marine oligosaccharide database, but also provide important basis for application in the field of medicine, as well as provide important reference value for other sugars with furan configuration.

海洋特征寡糖结构特异，生物活性广泛，在医药领域具备广阔的开发应用前景。发现并开发结构特异和活性独特的海洋特征寡糖仍然是海洋寡糖药物研究的重要方向。本课题组从一株深海真菌中分离得到一种结构特异，由长链呋喃半乳寡糖和甘露糖核心组成的半乳甘露聚糖。前期研究表明，该呋喃半乳寡糖具有显著的激活小鼠巨噬细胞的生物活性。为深入研究该类寡糖的结构特征和功能特性，本项目拟通过研究该类寡糖的二级质谱断裂规律，建立一种以ESI-MS/MS为主要手段的微量高效快速解析呋喃半乳寡糖的方法。探索呋喃半乳寡糖对巨噬细胞调节作用的机理，研究不同聚合度呋喃半乳寡糖激活巨噬细胞活性的强弱，获得最佳作用片段和浓度。从分子水平确定寡糖与巨噬细胞表面受体作用模式及对细胞因子IL-1β、TNF-α、NO的影响。本课题为首次系统的对呋喃半乳寡糖的结构和生物活性进行研究，为这类特征寡糖在医药领域的应用提供重要依据。

从采自太平洋2481m左右的深海海底底泥中分离得到的一株真菌灰黄青霉(Penicillium griseofulvum)的发酵液中分离得到一种结构新颖的半乳甘露聚糖。本课题以深海真菌胞外多糖半乳甘露聚糖为原料，首次对海洋微生物来源的特征呋喃寡糖进行研究。采用可控部分酸水解的方法制备结构特异的系列呋喃半乳寡糖，丰富了海洋特征寡糖库。在GC-MS和1D、2D-NMR技术表征寡糖的结构的基础上首次研究呋喃半乳寡糖在ESI-MS/MS的断裂规律和特征，建立以二级质谱为主的快速解析呋喃寡糖结构的新方法，为该类寡糖的结构鉴定和快速检测提供重要参考。. 国内外对呋喃半乳寡糖的活性研究较少。本课题对获得的不同聚合度的呋喃半乳糖进行巨噬细胞活性筛选，聚合度为4也就是呋喃半乳四糖的活性最高，能促进巨噬细胞的吞噬活力。呋喃半乳寡糖还能够被巨噬细胞识别内吞，能显著提高相关细胞因子的分泌。并首次发现呋喃半乳寡糖可以与半乳糖凝集素galectin-1相互作用，为进一步作用机制研究及构效关系研究提供深入资料，为进开发海洋微生物寡糖在药物方面的应用价值提供重要依据和物质基础。