基于免疫极化亲和色谱的中药多糖活性成分筛选研究

Macrophages are a type of antigen-presenting cells that engulf foreign substances in a process called phagocytosis. Macrophages can be polarized into two distinct phenotypes, including M1 (classically activated) and M2 (alternatively activated) macrophages, which are of great significance towards understanding the occurrence, development and treatment of diseases. Selecting polysaccharides from traditional Chinese medicine (TCM) as the object of study, this project constructs a novel immuno-polarized affinity chromatography (IPAC) derived from M1/M2 polarized macrophages for the first time, which will then be used to screen the immunoreactive polysaccharides from traditional Chinese medicine. The main contents of this project include the following points: construction of membrine receptor-based chromatographic stationary phase from M1/M2 polarized macrophages and characterization of its surface features, bioactivities and stability; investigation of IPAC-specific retention characteristics and peak area repeatability as well as the influencing factors; employing M1 and M2 polarized macrophages-based IPAC to identify the active ingredients of TCM polysaccharides and further screen the active monomers; evaluation of the immunoreactivity of polysaccharides and their specific immunoreactions (promoting/suppressing inflammation) via examining the expression of membrane surface molecules and immune factors; exploration of the correlations between the chromatographic retention behavior of polysaccharides and their structural properties, immunoreactivity and regulation of macrophage polarization; investigation of the in vivo immunological competence of the polysaccharides retained by the chromatographic columns, whereby to estimate the screening efficiency of this new type of IPAC. This project may provide a common platform for screening the immunoreactive ingredients like biological macromolecules and evaluating the polarization of macrophages.

巨噬细胞是机体内重要的吞噬和抗原提呈细胞，可极化为M1 和M2 两种表型，对免疫性疾病的发生、发展与治疗具重要意义。本项研究首次以中药多糖为研究对象，构建源于M1/M2巨噬细胞的新型免疫极化亲和色谱，并用于中药多糖免疫活性成分的筛选。主要内容包括：研制M1/M2巨噬细胞膜受体免疫极化色谱固定相，评价其表面特征、生物活性与稳定性；研究新型免疫极化色谱的保留特性及影响因素，考察其色谱保留重现性；分别运用M1、M2 型免疫极化亲和色谱确定中药多糖的活性部位，筛选活性单体成分；通过观测膜表面分子表达和免疫因子水平，比较评判多糖的免疫活性高低及极化特性（免疫促进/免疫抑制）；研究多糖色谱保留行为与多糖结构性质、免疫活性高低、极化调节特性之间的相关关系；考察色谱保留多糖的体内免疫活性，并籍此评价新型亲和色谱的筛选效能。本项研究可为生物大分子免疫活性成分筛选与极化类型研判提供新的共性平台。

重点围绕M1型和M2型多糖筛选、结构解析、体内外活性研究及构效关系展开系统研究，主要研究内容：1.成功构建了基于M1/M2巨噬细胞免疫极化的新型亲和色谱模型，并对免疫极化亲和色谱的系统性能进行了研究，结果表明，新型免疫极化亲和色谱专属性好，稳定性好；2.开展了M1型中药多糖免疫活性成分的筛选研究。以人参多糖为研究对象，运用M1 型免疫极化亲和色谱筛选具有M1极化活性的单体成分，人参粗多糖经纯化得到4种不同的纯化多糖GP-W-1，GP-0.1-1，GP-0.2-1和GP-0.3-1，经M1免疫极化色谱筛选，细胞及体内实验研究结果显示，人参多糖GP-0.3-1可促进巨噬细胞增殖、分泌NO及TNF-α，碳廓清指数及吞噬系数均显著上升，表明人参多糖GP-0.3-1具有M1极化活性；3.开展了M2型中药多糖免疫活性成分的筛选研究。以枸杞寡糖为研究对象，运用M2 型免疫极化亲和色谱筛选具有M2极化活性的单体成分，研究结果显示，枸杞寡糖LBO对巨噬细胞的碳廓清指数和吞噬系数均无显著影响，Western Blot实验结果显示，LBO可逆转M1型巨噬细胞成为M2型巨噬细胞，ELISA实验结果显示，LBO可显著促进巨噬细胞分泌IL-10，并抑制TNF-α及IL-β等炎症因子的产生，表明枸杞寡糖LBO具有M2极化活性；4.开展了中药多糖的构效关系研究。对人参多糖GP-0.3-1和枸杞寡糖LBO两种糖类进行结构鉴定，结果显示：GP-0.3-1主链由阿拉伯糖和半乳糖构成，侧链富含多种单糖，具有复杂的三股螺旋结构；而LBO仅由甘露糖和半乳糖构成，无三股螺旋结构；文献研究表明，甘露糖具有一定的抗炎活性，结合本项目研究结果，推测M2极化活性可能与多糖中甘露糖含量成正比，而M1极化活性则与甘露糖含量呈反比，且三股螺旋结构可增强多糖的M1极化活性。5.研究了枸杞寡糖LBO对脊髓损伤的修复效果。二代测序结果显示，LBO可增强外胚层间充质干细胞（EMSCs）的旁分泌功能，通过激活PI3k-Akt-mTor通路，诱导脊髓损伤部位的巨噬细胞的M2极化，可实现对脊髓损伤的修复。