松乳菇多糖（LDG-A)调控巨噬细胞免疫应答活性的分子机制

Polysaccharides are long carbohydrate molecules, of repeating units joined together bu glycosidic bonds. They are often linears, but may also be even highly branched. Polysaccharides are often quite heterogeneous, containing slight modifications od the repeating unit. Depending on the structure, these macromolecules can have distinct properties from their monosaccharide building blocks. They may be amorphous or even insoluble in water. Polysaccharides known as "biological response modulators", reflects its significant immunomodulatory activity, such as promoting the proliferation and differentiation of immune cells, activated T cells and B cells, secrete a variety of lymphokines, regulate the balance of nerve-endocrine-immune regulatory network, and promote the DNA, RNA, protein synthesis, et al..In recent years, as more and more polysaccharides have been reported to exhibit a variety of biological actvities, including anti-tumor, immunostimulation, anti-oxidation etc., the nonstarch polysaccharides have emerged as an important class of bioactive natural products. In many oriental countries, several immunoceuticals composed of polysaccharides have been accepted such as lentinan, schizophyllan and krestin. Stimulation signal transduction of polysaccharides in the macrophages are mainly involved activation of NF-KB region and MAKPs region. At the same time, polysaccharides can stimulate Ca2+ influx in the macrophages, and showed similar effects of PKC activator, which may involve activation of NF-kB region and MAPKs region, but the mechanism is still not clear. A novel heteropolysaccharide (LDG-A) was isolated from the fruiting bodies of Lactarius deliciosus (L. ex Fr.) Gray in our previous study. LDG-A can enhance the phagocytosis of macrophage (RAW264.7) in vitro. Further research showed that LDG-A polysaccharide has an important effect on immune factors of macrophages in transcription and translation level, the immune response in macrophage of LDG-A are involved activation of NF-kB and MAPKs signal transduction pathway of the signal transduction involved NF-kB and MAPK s region. This study aimed to explore the signal transduction NF-kB and MAPK s signal transduction pathway of LDG-A in macrophage at the cell level; to further explore the relationship between Ca2+ influx and NF-kB and MAPKs signal transduction pathway for further investigation of its mechanism for the theoretical foundation and application mode.

多糖多具有抗氧化、抗肿瘤和免疫调节等生物学活性。多糖刺激巨噬细胞内的信号转导主要涉及NF-κB区和MAKPs区的激活。同时，多糖能刺激巨噬细胞中的Ca2+ 内流，与PKC等激活剂表现出相似的作用，从而可能涉及NF-κB区及MAPKs 区的激活，但该机制仍不明确。本课题组首次从松乳菇子实体中分离得到纯品多糖LDG-A，体外具有增强巨噬细胞（RAW264.7）吞噬的能力，进一步研究显示松乳菇多糖（LDG-A）对巨噬细胞相关免疫因子的转录和翻译水平具有重要影响，其调控巨噬细胞免疫应答信号通路涉及NF-κB区和MAKPs区的激活。本项目拟在分子水平进一步探寻松乳菇多糖（LDG-A）调控巨噬细胞所涉及的NF-κB 通路与 MAPKs 通路的信号转导情况，并证实该多糖与细胞内钙及其可能涉及的NF-κB信号转导与 MAPKs 信号转导通路的关系，以深入研究其机制。

多糖多具有抗氧化、抗肿瘤和免疫调节等生物学活性。多糖刺激巨噬细胞内的信号转导主要涉及NF-κB区和MAKPs区的激活，但该机制仍不明确。本课题组首次从松乳菇子实体中分离得到纯品多糖LDG-A，本项目通过三年的研究工作，进一步确认了松乳菇多糖 (LDG - A) 对S180荷瘤小鼠生理生化指标的影响，显示松乳菇多糖LDG-A对小鼠的肝脏无毒副作用，且能缓解S180荷瘤小鼠的肝脏损害，同时松乳菇多糖LDG-A不影响小鼠的脂肪代谢途径。完成了对照组和LDG-A组之间巨噬细胞转录组差异的比较，利用Cat # QAM-CAA-4000芯片分析了LDG-A对巨噬细胞200个细胞因子的影响，证实LDG-A在体内的显著的抗肿瘤活性涉及激活巨噬细胞的MAPK和NF-κB信号通路。完成了对巨噬细胞RAW264.7细胞细胞周期分析，LDG-A可通过阻断G2 / M期细胞周期阻滞，促进G0 / G1期细胞周期进程，从而诱导细胞分裂，促进巨噬细胞的增殖。检测了LDG-A刺激下，巨噬细胞吞噬活性及对L929肿瘤细胞的抑制活性的变化。完成了激光共聚焦下巨噬细胞骨架的观察。应用Western blot 技术检测了正常对照组和 LDG-A 实验组及采用NF-κB抑制剂后巨噬细胞的 NF-κB 信号通路中关键蛋白的变化。应用Western blot 技术检测了正常对照组和 LDG-A 实验组及采用PKC抑制剂后巨噬细胞的 MAPKs 信号通路中关键蛋白的变化。应用ELISA技术检测了LDG-A诱导下蛋白分泌情况。通过以上研究，使我们对松乳菇多糖（LDG-A）调控巨噬细胞所涉及的NF-κB 通路与 MAPKs 通路的信号转导情况有了更深入的认识。.此外我们对松乳菇多糖LDG-A调控特异性免疫细胞增值和肿瘤细胞凋亡的分子机制进行了广泛的研究，包括T细胞，B细胞， A549细胞，Hep-2细胞等，同时对松乳菇多糖LDG-B和寡聚糖LDGO-A的结构，药理活性，分子机制和构效关系进行了初步探寻。在该项目的资助下，本研究团队对中国西南地区食药用真菌及其活性代谢产物的进行了筛选，并对代谢产物活性及其分子机制进行了广泛的研究，先后申请多项国家发明专利。.以上研究数据为多糖类靶向免疫调控剂的开发提供了一定的科学依据。