tlr4受体介导的壳寡糖对巨噬细胞激活机制的分析-analysis of activation mechanism of chitooligosaccharides on macrophages mediated by tl r4 receptor.docxVIP

巨噬细胞不同时间后，荧光显微镜观察结果表明巨噬细胞能够吞噬壳寡糖，随着时间的延长细胞吞噬壳寡糖的量不断增加，在 15min 左右达到吞噬高峰； TLR4 单克隆抗体预处理巨噬细胞 1h 后再加入 FITC-COS，巨噬细胞对壳寡糖 的吞噬作用几乎完全被抑制。壳寡糖能促进 RAW264.7 细胞的增殖，提高其吞 噬中性红的能力，以及分泌 NO 的能力，各类寡糖糖样中以壳六糖的效果最好。 TLR4 单克隆抗体预处理细胞后再加入 COS，RAW264.7 细胞分泌 NO 的能力急 剧降低。壳寡糖能够诱导巨噬细胞内 TNF，iNOS，TLR4 等基因转录，但结果 仍需进一步探讨。结论：壳寡糖（3~7 聚合度）能够被巨噬细胞吞噬，进而激活巨噬细胞， 具有较好的体外、体内免疫调节功能，壳寡糖对巨噬细胞的激活是通过细胞表 面TLR4 受体参与介导的。关键词： 壳寡糖；FITC；巨噬细胞；免疫功能；TLR4Research on the mechanisms of macrophage activationby chito-oligosaccharides mediated by TLR4 receptor AbstractBackground: Chito-oligosaccharides, the degration product of chitosan, has more valuable biological activities than Chitosan such as enhance immunity, anti-tumor, antioxidant, antimicrobial, promoting healing and so on, which is due to its feature of low molecular weight, non-toxic and good water solubility. There’s study showed that chito-oligosaccharides can regulate immune function of the organism and inhibit tumor cell from growing and metastasis. Our work provides a foundation for further research of immunoregulation mechanism of oligochitosan with macrophage.Methods: Chitosanase was used to prepare chito-oligosaccharides through the enzyme hydrolysis method in this project ,and HPLC method was used to identify the composition of COS. The effects of different concentrations of COS on the proliferation, neutral red phagocytosis ability, and tumor necrosis factor (TNF-alpha) secretion of mouse peritoneal macrophages were tested in vitro studies. Mice were administrated with different doses of COS to elucidate the impact of COS on serum IgG and IgM levels and on the thymus, spleen index of mouse. COS was then labeled with fluorescein isothiocyanate (FITC) to get FITC-COS. Phagocytosis of COS by mouse peritoneal macrophages and its relationship with TOLL-like receptor 4 (TLR4) was investigated.Results: HPLC analysis results showed that COS, prepared by chitosanase hydrolysis, mainly consist of 3-7 degree of polymerization of monosaccharide. Chitosan has prefer