发菜的多糖免疫调节功能及其分子机制

Nostoc flagelliforme is one kind of terrestrial cyanobacteria grown in the arid and semi-arid areas. After years of research efforts, the artificial cultivation technology of the Nostoc flagelliforme cells combined with the production of polysaccharide in China reached lead level of the world. The study found that Nostoc flagelliforme polysaccharides are heteropolysaccharides composed of a variety of monosaccharides and glucuronic acid with rare special structure of non-sulfated acidic polysaccharides. In recent years they are found to have anti-virus, anti-tumor and immune regulatory function. However, how many special immune regulatory functions they have and the relation with the structure particularity of their polysaccharide are unclear. Even more, whether there are any new immune mechanisms are unknown. The lack of basic knowledge of their nature, especially immune regulation function and mechanism seriously holds back the further development and application of Nostoc flagelliforme polysaccharides, therefore, this project starts with the analysis and comparison of the compositions and structures of the polysaccharides from different origins (wild, autotrophic and mixtrofic),further, their basic immune functions wiil be examined from the effects on macrophage, T and B cells.Subsequently.The effect of N.flagelliforme polysaccharides on mRNAs of cytokines will also be investigated. In addition, the impact of N.flagelliforme polysaccharides on changes of GSH, GSSG,cAMP、 cGMP,Ca2+ and NO levels are planned to be studied from the level of signal transduction. Furthermore, the single-molecule force spectroscopy，SMFS is emploied to investigate the interaction and conformation differences between the molecules with different origins and the immune cell surfaces. On the basis of these fundamental studies, the QSAR will be built between the structure and the the immune regulation functions of N.flagelliforme polysaccharides. With this research, not only further understanding of the polysaccharide immune mechanism can be extended, but also the development of polysaccharide products with unique immune features and characteristics will be promoted. This study has an important role for the maintaining the lead level of our country in the artificial cultivation and application of Nostoc flagelliforme.

发菜（发状念珠藻）为生于干旱半干旱地区的国家珍稀陆生蓝藻，其多糖为非硫酸酸性杂多糖，近年来初步发现其免疫调节功能。其独特组成和结构如何影响免疫系统功能以及不同来源的（野生和人工培养）发菜多糖生物活性存在差异性问题值得进行深入研究。因此，本项目首先对不同来源的发菜多糖组成和结构进行比较分析，再进一步从其对巨噬细胞、T细胞和B细胞及细胞因子的影响考察其基本免疫功能，在基因水平考察发菜多糖对巨噬细胞因子mRNA表达影响，并从信号转导水平考察发菜多糖对小鼠淋巴细胞内GSH、GSSG、cAMP、cGMP、Ca2+和NO水平变化的影响。同时，利用单分子力谱考察多糖与免疫细胞相互作用差异及构象变化，在此基础上，建立多糖成分、结构对免疫调节功能的构效关系，获得对多糖免疫功能及机制的系统认识。该研究对于促进人工培养发菜多糖结构改造和应用开发，保持我国在该物种人工培养领域的领先优势具有促进作用。

发菜（发状念珠藻）为生于干旱半干旱地区的国家珍稀陆生蓝藻，为国家一级保护植物。近年来研究表明，发菜细胞通过人工液体培养，可获得比野生状态下高1000倍以上的生长速度，且可同时从培养液中获得胞外多糖。发菜胞外多糖为非硫酸酸性杂多糖，具有抗肿瘤、抗病毒、抗氧化和抗疲劳活性，并初步发现其免疫调节功能。其不同来源（野生和人工培养）发菜多糖组成和结构有何差别？对免疫系统功能影响有何差别值得进行深入研究。因此，本项目首先对不同来源的发菜胞外多糖的组成、结构和物理性质进行了比较分析，发现野生发菜多糖组分EPSA-1和人工自养发菜多糖组分EPSB-1和混养发菜多糖组分EPSC-1均由9个单糖和糖醛酸组成，野生发菜多糖组分EPSA-2则不含脱氧糖，由7个单糖和糖醛酸组成。. 对野生和人工自养发菜多糖进行了体内和体外免疫活性研究。分别试验了无刺激物、刀豆蛋白A（ConA）和脂多糖(LPS)丝裂原诱导三种条件下发菜多糖的免疫调节活性。尽管野生和人工自养培养发菜多糖各种免疫活性有所差别，但总体来说，它们对小鼠脾淋巴细胞增殖、NK细胞活性和单核巨噬细胞、腹腔巨噬细胞吞噬能力均有一定促进作用。它们都能够提高小鼠血清中细胞因子IL-6、IL-12浓度。体内免疫实验显示发菜多糖正常小鼠体重和脏器指数影响不大，发菜胞外多糖对小鼠无毒副作用。该研究为进一步从分子水平、基因水平、信号转导水平上阐明发菜多糖免疫调节机制奠定了基础。该研究对于人工培养发菜多糖结构改造和应用开发，保持我国在该物种人工培养领域的领先优势具有促进作用。. 通过本项目研究，发表SCI论文1篇，另有1篇SCI论文被接受，申请国家专利2项，获得国家发明专利授权1项， 毕业硕士研究生1人。.