海参糖胺聚糖通过TLR4受体促进巨噬细胞功能的分子机制及联合环磷酰胺抗乳腺癌的作用研究

As the nourishing food, sea cucumber exhibits excellent anti-tumor and immune-enhancing properties. Fucosylated chondroitin sulfate (fCS) is the main bioactive component in sea cucumbers. We obtained three kinds of fCSs from sea cucumber Apostichopus japonicus, Stichopus chloronotus and Acaudina molpadioidea, and the structural difference mainly lies in their fucose branches. Our previous studies indicated that fCSs could activate macrophage functions via Toll like receptor 4 (TLR4) and NF-κB signaling pathway, and the effects of different fCSs are significant. In order to further explore the mechanism of immunoregulation of fCSs, the RT-PCR method, western blot assay and PCR array analysis will be employed to detect the receptor types of fCSs and intracellular signaling pathway on the Raw264.7 cells. The confocal laser scanning microscope will be used to determine the combination potential of 2-AMAC labeled fCS and the receptors. Furthermore, the cyclophosphamide induced immunosuppression mouse model and breast cancer mouse model will be recruited to investigate the chemoprotective effects and the anti-breast cancer effects of fCSs combined with cyclophosphamide. The molecular mechanisms of activation properties of fCSs on macrophage, their chemo-protective and anti-cancer effects will be firstly investigated in our project, which would benefit the understanding of the immune-regulatory activities of fCSs and the development of new immune-enhancement drugs.

海参作为滋补食品，被认为有抗肿瘤和提高免疫力等功效。海参糖胺聚糖（fCS）是其中主要的活性多糖。我们前期从不同海参中分离出三种fCS，其结构差异主要在于硫酸化的岩藻糖支链。活性实验表明，fCS通过Toll样受体4（TLR4）及NF-κb信号通路激活巨噬细胞功能，且不同fCS的作用差异显著。为深入探讨fCS的免疫调节作用机制，本课题将以小鼠单核巨噬细胞Raw264.7为模型，运用QPCR、免疫印迹及PCR阵列等技术检测细胞表面能识别fCS的TLR类型及fCS激活的胞内信号转导通路；运用共聚焦显微镜观察细胞对荧光标记fCS的识别和结合能力；利用免疫抑制和荷瘤小鼠模型，研究fCS增强免疫力和联合环磷酰胺抗乳腺癌的作用。本研究首次从分子水平阐明fCS激活巨噬细胞的功能和构效关系及其提高免疫力和联合化疗药抗乳腺癌的作用，为深入研究其免疫调节活性，开发海参糖胺聚糖类免疫增强药物提供理论依据。

海参是一种名贵的食品和中药材，具有免疫调节和抗肿瘤等功效，海参糖氨聚糖（fCS）是其主要活性成分，但是fCS的免疫调节和抗肿瘤作用机制仍不明确。本项目以课题组前期分离纯化的三种海参糖氨聚糖为原料，采用HPSEC-MALLS-Vis-RI检测法、Worm-like cylinder模型及多种理化分析方法对其构象和物理性质进行了测定；以小鼠单核巨噬细胞Raw264.7为模型，运用荧光标记-共聚焦显微镜观察、QPCR、免疫印迹等技术检测细胞表面能识别fCS的TLR类型及fCS激活的胞内信号转导通路；利用免疫抑制小鼠模型，研究fCS抵抗环磷酰胺导致的免疫抑制的作用。结果表明，仿刺参糖氨聚糖（fCS-Aj）呈现无规则卷曲的链状构象，而北极参糖氨聚糖（fCS-Hm）和绿刺参糖氨聚糖（fCS-Sc）呈刚性的棒状结构，三种糖胺聚糖均为聚阴离子体，且具有较高的热稳定性；通过活性筛选获得活性最优且结构新颖的绿刺参糖氨聚糖（fCS-Sc），fCS-Sc能够通过TLR2/4-NF-κB信号通路激活巨噬细胞，促进其增殖和吞噬能力，及分泌NO、TNF-α、IL-1β、IL-6的能力，增强巨噬细胞的免疫功能；此外，fCS-Sc能缓解环磷酰胺导致的小鼠脾指数降低，具有潜在的抗免疫抑制作用。本研究首次从分子水平阐明fCS激活巨噬细胞的功能及其提高免疫力的作用，为深入研究其免疫调节活性，开发海参糖胺聚糖类免疫增强药物提供理论依据。