利用真菌作为降解工具研究果胶的结构与免疫活性的关系

Pectin is one of the most complex biomacromolecules, which is consisted of various structural units or domains. These domains are important to pectin function and activity. In this project, the pathogenic fungi would be used as the tool for degradation to prepare series of pectin domains, then, the immunological activities of obtained pectin domains would be tested to explore the structure-activity relationship. This study includes: 1) Fungi with pectin-degradation activity will be primarily screened by Congo red staining with ginseng pectin as the substrate in solid medium followed by liquid medium as well as HPLC detection, the strains with stable pectin-degradation activities would be obtained, and the degradation conditions would be optimized. 2) 10-15 kinds of pectins from different plants would be degraded by screened fungi, the degradation products would be isolated and the structure characters would be analyzed. 3) The effects of different pectin domains on the differentiation, maturity, proliferation and activation of immune cells DC, NK, αβT and γδT would be systematically studied, and the relationship of structures and activities would be deduced. This project will provide new ideas for exploring the relationship of structure-activity in pectin, and the results would enrich the theory of glycobiology. Since the immune cells used in this projects are the ones commonly used in tumor immunotherapy and were proved to have remarkable effects, therefore, the results would also explore novel direction for the pharmacological application of pectin.

植物果胶是多种结构单元或结构域共价连接形成的生物大分子，这些结构单元对果胶的功能和活性有重要的影响。本项目利用植物病原真菌作为降解果胶分子的工具，制备系列结构单元，进而研究它们的免疫活性，探讨结构与免疫活性的关系。研究内容包括（1）以人参果胶为底物，固体培养，刚果红染色法检测，初步快速筛选可降解果胶分子的真菌；然后液体培养，高效液相检测复筛，确定能够稳定降解果胶的菌种，并优化降解条件。（2）用筛选出的植物病原真菌和优化的条件降解10-15种植物果胶，分离纯化降解产物，分析结构特征。（3）研究结构不同的系列果胶分子对免疫细胞DC、NK、αβT和γδT的分化、成熟、增殖、活化等影响，探索结构与免疫活性的关系。本项目为探索果胶结构与活性的关系提供了新的思路，研究结果丰富了糖生物学理论。由于采用的免疫细胞是肿瘤患者免疫治疗中常用的细胞，具有明确疗效，因此，研究成果也为果胶的实际应用开辟了新的途径。

果胶是自然界中结构最为复杂的一类生物大分子，具有多种生物学活性。不同结构的果胶活性差异较大，因此制备不同的果胶结构域对于研究果胶的活性及其构效关系具有重要意义。本项目利用微生物作为降解工具，制备了不同类型的果胶结构域，研究了它们的结构特征，并探讨了果胶结构与免疫活性的构效关系。首先以人参果胶为底物，经过固体培养和液体培养，筛选出一系列可稳定降解果胶的微生物，优化了降解条件，并建立了稳定的微生物转化系统。接着利用这些微生物降解了人参、罗布麻、藜芦、槲寄生、玄参、党参等不同来源的植物果胶，制备出近50种不同的果胶结构域及其分子片段。综合运用酸水解、酶解、核磁共振及质谱的手段分析这些结构域的特征，证明不同微生物降解果胶的产物主要为RG-II结构域、RG-I结构域及HG结构域，补充了实验室现有的果胶分子库。进一步利用体外细胞实验分析不同果胶结构域对NK细胞和γδT细胞增殖活化的影响，结果表明不同类型果胶的免疫活性不同，RG-I型果胶结构域具有促进免疫细胞增殖的活性，优于HG和RG-II结构域。本项目的研究结果为果胶结构域的制备提供了新的手段，为果胶免疫活性的构效关系研究提供了新的数据，对多糖的理论研究和实际应用都具有重要的意义，对糖生物学发展起到积极的促进作用。项目执行期间共发表25篇SCI论文，参加国际国内会议12次，获得授权发明专利2项，培养博士研究生6名，硕士研究生11名。