用酶解法制备壳寡糖及其对机体免疫功能的调节作用  

Modulation of immunol-properties by chito-oligosaccharide

作　　者：张沛[1] 韩宝芹[1] 陈列欢[2] 彭燕飞[1] 刘万顺[1] 郑汉东[1] 杨艳[1] 

机构地区：[1]中国海洋大学海洋生命学院,青岛266003 [2]江苏省淮海工学院海洋学院,连云港222000

出　　处：《中国免疫学杂志》2013年第2期191-196,共6页Chinese Journal of Immunology

基　　金：山东省青岛市科技项目支持[12-1-4-1-(23)-jch]

摘　　要：目的:研究本课题组酶解制备的主要含3-7聚合度壳寡糖(COS)对机体免疫功能的调节作用。方法:利用本课题组分离的高活性壳聚糖酶通过酶水解法制备壳寡糖,HPLC法对壳寡糖的成分进行鉴定,并用异硫氰酸荧光素(FITC)将壳寡糖进行荧光标记得到FITC-COS,研究小鼠腹腔巨噬细胞对壳寡糖的吞噬作用及其与Toll样受体4(TLR4)的关系,进一步研究了不同浓度壳寡糖对小鼠腹腔巨噬细胞的增殖,吞噬中性红能力及分泌TNF-α能力的影响,并对小鼠灌胃不同剂量的壳寡糖,研究了壳寡糖对小鼠血清IgG和IgM含量及小鼠胸腺、脾脏指数的影响。结果:HPLC分析结果显示壳聚糖酶水解法制备的COS主要为3-7单糖聚合度的寡糖。将FITC-COS作用于小鼠腹腔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COS,随着时间的延长吞噬COS的量增加,TLR4单克隆抗体预处理巨噬细胞1小时后再加入FITC-COS,巨噬细胞对COS的吞噬作用几乎完全被抑制。COS被巨噬细胞吞噬后可显著增强巨噬细胞的吞噬功能,刺激巨噬细胞分泌TNF-α。体内研究结果表明小鼠灌胃COS能够显著增加小鼠的脾脏指数,增加血清中IgG的含量,对胸腺指数和血清中IgM的含量没有显著影响。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受体介导的。Objective：To study the immunol- regulation properties of chito-oligosaccharides （COS） with 3-7 degree of polymer- ization, which are prepared by enzymatic hydrolysis. Methods：Chitosanase founded by our research group was used to prepare chito-oli- gosaccharides through the enzyme hydrolysis method, and HPLC method was used to identify the composition of COS. COS was then la- beled with fluorescein isothiocyanate （FITC） to get FITC-COS. Phagocytosis of COS by mouse peritoneal macrophages＇ and its relation- ship with TOLL-like receptor 4 （ TLR4 ） was investigated. The effects of different concentrations of COS on the proliferation, neutral red phagoeytosis ability, and tumor necrosis factor （TNF-alpha） secretion of mouse peritoneal maerophages were then tested. 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. Results ： HPLC analysis results showed that COS, prepared by chitosanase hydrolysis, mainly consist of 3-7 degree of polymerization of monosaccharide. Fluorescence microscopy results showed that FITC-COS could be phagocytized by mouse peritoneal macrophages in a time-dependent manner. Pretreatment of maerophages with TLR4 monoelonal antibody for l hr, phagoeytosis of COS was almost completely inhibited. The phagocytosis and TNF-c~ secretion abilities of macrophage were all significantly enhanced after COS was phagocytized. The resuhs of in vivo studies showed that administration of mice with COS could significantly increase the spleen index and serum IgG levels. While the thymus index and serum IgM levels were not significantly changed. Conclusion ： COS with 3-7 polymerization degree can be phagocytized by macrophage and then activate macrophages. COS has good in vitro and in vivo immu- nological function. The activation of macrophages by COS is mediated by the TLR4 receptor on cell surface.

关 键 词：壳寡糖 FITC 巨噬细胞 免疫功能 

分 类 号：R392.12[医药卫生—免疫学]