粒毛盘菌属多糖结构、分子修饰与生物活性研究

Fungal polysaccharides have a variety of biological activities. In preliminary studies，we found that the antitumor activity of Lachnum extracellular polysaccharide (LEP-1a) had increased significantly after being phosphorylated modification. In this project, we will intend to carry out supplementing points collection, identification and classification for samples to expand germplasm resources in Southwest China and other regions. Eight to ten high-yield of polysaccharides strains will be selected and fermented to separation extracellular polysaccharides. Then the strength of the antitumor, anti clotting, anti chronic renal failure and hypolipidemic activities of the polysaccharides will be evaluated. The polysaccharides with strong activities will be fractionated and their activities will be tracked respectively. And highly active Lachnum extracellular polysaccharides (LEP) will be chosen to analyze their structural characterization using mass spectrometry, infrared spectrum, nuclear magnetic resonanceand so on, and modified by phosphorylation, sulfation, carboxymethylation, benzoylation modification methods respectively. The strength of the antitumor, anti clotting, anti chronic renal failure and hypolipidemic activities of 4-6 strong activie polysaccharide derivatives selected will be evaluated. Through this research, 3-4 kinds of LEP modification methods will be established. And the structure-activity relationship of LEP will be clarified and hypolipidemic mechanism will preliminarily be revealed, providing the scientific basis for the deep development and utilization of LEP resources. This study is expected to obtain new polysaccharides and their derivatives with independent intellectual property rights.

真菌多糖具有多种生物活性。前期我们研究发现，粒毛盘菌胞外多糖LEP-1a经磷酸化修饰后其抗肿瘤活性显著提高。本项目拟赴西南等地区进行粒毛盘菌属标本的补点采集、分类鉴定与菌株分离，以扩充种质资源；选择8-10株高产多糖菌株进行发酵、分离胞外多糖，进行抗肿瘤、抗凝血、抗慢性肾衰竭、降血脂活性筛选；选择活性较强的多糖进行分级分离与活性追踪，继而采用质谱、红外光谱、核磁共振等技术对其进行结构解析，并分别进行磷酸化、硫酸化、羧甲基化、苯甲酰化修饰，筛选4-6个高活性多糖衍生物进行抗肿瘤、抗凝血、抗慢性肾衰竭、降血脂活性研究。通过本项目研究，建立3-4种粒毛盘菌多糖修饰的方法，阐明粒毛盘菌多糖的构效关系，初步揭示粒毛盘菌多糖降血脂作用机理，为粒毛盘菌属多糖资源的深度开发与利用提供科学依据。本研究有望获得具有自主知识产权的新多糖及其衍生物。

本项目赴西南等地区补点采集了粒毛盘菌属标本50余个、分类鉴定出20余株粒毛盘菌，筛选出6株高产菌株发酵提取胞外多糖，选择了5个活性较强的多糖进行分级分离，通过凝胶层析技术制备均一性多糖。采用红外光谱、核磁共振等技术对其进行结构解析，共解析出5个粒毛盘菌多糖结构，并分别使用硫酸化、羧甲基化、二肽修饰等方法修饰多糖，筛选出高活性多糖衍生物进行抗肿瘤、抗慢性肾衰竭、降血脂、降血脂、抗疲劳、抗凝血、抗氧化及免疫等活性研究。部分结果如下：..粒毛盘菌YM240发酵液经水提醇沉、脱色和脱蛋白后获得胞外多糖，再经DEAE-cellulose 52和Sephadex G-100色谱柱分离纯化，得单一组分LEP；HPLC、GC-MS、甲基化分析、FT-IR和1D/2D NMR等实验解析得到YM240的结构。对LEP进行羧甲基化、硫酸化修饰及二肽修饰，结果表明与粒毛盘菌多糖相比，修饰后得到的多糖衍生物的降血糖、降血脂及抗氧化活性明显提高。此外，粒毛盘菌多糖二肽修饰衍生物的理化性质明显得到了改善，免疫调节活性也得到提高。..体外抑瘤实验结果表明， YM130中提取的多糖LEP-2a与5-FU具有显著的协同作用。Western blotting结果表明，LEP-2a与5-FU联合处理能够抑制Ras/Raf/MEK/ERK 及 PI3K/AKT/mTOR信号途径，抑制由Nrf2-keap1介导的抗氧化防御系统、增加细胞中活性氧的表达并引起线粒体凋亡途径的激活。联合处理能够使HepG-2细胞周期停滞在S期，使5-FU作用于细胞的时间延长；LEP-2a还能抑制DNA错配修复及DNA损伤修复，从而抑制了细胞损伤的修复。体内抑瘤（H22）实验结果表明， LEP-2a与CTX具有显著的协同作用。免疫组化、免疫荧光、Western blotting、组织病理学分析、ELISA等结果表明，LEP-2a与CTX的协同抗H22肿瘤作用主要通过Fas/FasL介导的半胱天冬蛋白酶依赖性死亡途径以及线粒体凋亡途径实现的。研究发现LEP-2a能够显著提高机体免疫应答，抑制肿瘤中血管生成及抑制细胞存活途径，并能显著缓解由CTX引起的机体免疫抑制、氧化应激、肝肾损伤等副作用。.通过本项目实施，共发表SCI论文17篇，申报国家发明专利5项，2项获得授权，培养青年教师1名，硕士研究生5名、博士研究生2名（1名境外博士生）。