【作者】 徐文清；

【导师】 高文远；

【作者基本信息】 天津大学 ， 应用化学， 2006， 博士

【摘要】 目的:随着糖生物学研究的深入,多糖的结构及活性研究取得了积极的成果,多糖具有多方面的生物活性。银耳(Tremella fuciformis Berk),是一种高等真菌,是世界公认的珍贵食用菌和重要药材。为了探索银耳中活性成分的构效关系,本文对银耳孢子发酵粉中的多糖成分进行了提取、分离及结构分析,考察其抗肿瘤、保护造血功能、增强免疫等生物学活性,用基因表达谱研究抗肿瘤作用的机制。以期为银耳资源的开发奠定基础。方法:采用DEAE-SephadexA-25和SephadexG-150对银耳孢子发酵粉中的水溶性多糖进行了分离、纯化,得到两个均一体银耳孢子多糖,WTF-A和WTF-B。采用DEAE-32-Cellulose和Sephadex G-200对碱溶性多糖进行了分离、纯化,得到均一体银耳孢子多糖,ATF。通过化学和光谱学方法,分析了三个多糖的结构特征。对WTF-B进行抗肿瘤、保护受辐射和化学损伤小鼠的造血功能及增强免疫功能的研究,用基因表达谱研究WTF-B抗肿瘤作用的机制。结果:WTF-A主要由1,6连接的半乳糖组成;WTF-B主要由1,4连接的葡萄糖和葡萄糖醛酸组成;ATF主要由1,6连接的葡萄糖组成。小鼠体内抗肿瘤实验结果表明,WTF-B单独及合用γ射线照射,对多种瘤谱有很好的抑制作用。WTF-B对辐射和化学损伤小鼠造血系统影响的实验表明,对7.5 Gy 137Cs-γ射线照射引起的小鼠造血功能损伤,可使受照小鼠的股骨有核细胞数、脾指数和脾结节明显增加。对环磷酰胺和阿糖胞苷引起的小鼠造血功能损伤,银耳孢子多糖可以拮抗化疗药引起的白细胞降低。WTF-B对免疫功能的影响实验表明,银耳孢子多糖可以显著地提高免疫功能低下小鼠的非特异性免疫和特异性免疫,对正常小鼠的免疫功能无明显影响。用基因表达谱研究了银耳孢子多糖WTF-B对荷H22肝癌小鼠抑制作用的机制。共有324个基因有表达差异。其中表达上调基因185个,表达下调基因139个。这些表达差异基因分为信号转导基因,细胞因子、转录因子基因,细胞基质和细胞粘连相关基因,转运和泵基因,p53上游信号/p53调节剂基因,p53信号通路基因,DNA损伤检测/p53和ATM通路基因,G1期基因,干细胞和分化细胞的分子标志基因,乳腺癌直接相关基因,抗原呈递基因,化疗应答相关基因等。结论:首次阐明银耳孢子多糖WTF-A、WTF-B和ATF三种新结构杂多糖的结构特征。WTF-B对多种瘤株具有较强的抑制作用,可以保护受辐射和化学损伤小鼠的造血功能,提高免疫功能低下小鼠的免疫力。抗肿瘤作用机制是多靶点﹑多因素作用的结果,既与癌症相关基因﹑又与免疫调节﹑信号转导等基因有关。更多还原

【Abstract】 Objective: With development of the research about Carbohydrate Chemistry andGlycobiologyin latest years,a great ofachievements about the study onthe structureand activity of polysaccharides have been made. Polysaccharides exhibit manybiological activities,including of anticancer, anti-virus, anti-infection, radiationprotection, immunological regulation, promoting biological synthesis of protein andnucleic acid. Tremella fuciformis Berk belongs to tremellaceae of Heterobasidio-mycetae, it is known as nutritious mushroom and important medicine by people allover the world. To explore the relationship between structure and bioactivity ofpolysaccharide, this paper presents the extraction, isolation, identification ofpolysaccharides from spores of Tremella fuciformis Berk fermentation products, andinvestigates its bioactivities of anti-tumor, hematopoietic protection, immuneenhancement and mechanism of inhibition to H22 hepatoma in the way of cDNAmicroarray.Methods: DEAE-Sephadex A-25 and Sephadex G-150 were adopted toseparate andpurify the water solution polysaccharides from spores of Tremella fuciformis Berk.Two homogeneous polysaccharides named WTF-A and WTF-B have been obtainedand identified. DEAE-32-Cellulose and Sephadex G-200 were adopted to separateand purify the alkaline solution polysaccharides, a homogeneous polysaccharidesnamed ATF has been obtained and identified. The structure feature of WTF-A,WTF-B and ATF were analyzed by chemical methods as whole hydrolyzed, partialhydrolyzed, methylated and spectroscopic methods as UV, IR, GC, GC-MS, NMR.The effects on anti-tumor, hematopoietic protection, immunologic enhancement ofWTF-B were performed. Its mechanism of inhibition on H22 hepatoma of WTF-Bwasalsoexploredbywayof cDNAmicroarray.Results: WTF-A is composed of galactose, glucose, mannose and two heptoses incloselymolar ratios of 7:5:1:1:1. Its molecular weight is estimatedtobe73000byHPGPC. Its main chain is comprised of 1,6 linked galactose and 1,2,3,4,6 linkedmannose or 1,3,6 linked mannose. Side chain are comprised of terminal glucose,terminal mannose, 1,2,4 linked mannose, L-glycero-D-glucoheptopyranoside andL-glycero-D-mannohepopyranoside.WTF-Bis composedofglucose,mannoseandserineincloselymolarratios of8:2:0.1.Itsmolecularweightis estimatedtobe68000byHPGPC.Itsmainchainiscomprisedof 1,4 linked glucose and 1,2,3,4,6 linked mannose, the branch point isat the site ofmannose. Side chain are comprised of terminal glucose, Serine, H2N-1,2,3 linkedmannose.Glucoseuronicacid maybeinmainchainbecauseofits 32.88%content.更多还原