广叶绣球菌中性多糖对巨噬细胞RAW 264.7细胞因子及TLR2受体的影响

Effects of Sparassis latifolia Neutral Polysaccharides on Cytokines and TLR2 Receptor of Macrophage RAW 264.7

真菌多糖主要通过TLRs受体介导吞噬细胞分泌细胞因子而进行免疫调节作用,为探究广叶绣球菌中性多糖(SNP)对巨噬细胞RAW 264.7细胞因子的分泌及TLR2受体是否介导此作用,首先采用传统水提法,经HZ-830大孔树脂、DEAE-52和Sepharose CL-6B柱分离纯化后得到SNP,利用高效凝胶渗透色谱法(HPGPC)测定SNP分子质量,利用离子色谱法(IC)和傅里叶红外色谱法(FT-IR)分析单糖组成和结构表征;然后通过MTT比色法分析SNP对巨噬细胞RAW 264.7增殖活力的影响,以SNP最佳浓度作用,通过酶联免疫吸附测定法检测巨噬细胞RAW 264.7分泌的细胞因子含量;最后通过SNP处理TLR2抗体作用的吞噬细胞,分析TLR2受体是否对SNP调节巨噬细胞RAW 264.7细胞因子的分泌具有介导作用。结果表明,SNP分子质量为3.2×10;u,由摩尔比为6∶12∶63∶10∶5的阿拉伯糖、半乳糖、葡萄糖、木糖、甘露糖组成,不含呋喃糖;一定浓度范围内的SNP能显著促进RAW 264.7的增殖,其最佳作用质量浓度为250μg/mL;此浓度下,RAW 264.7的NO、IL-6、TNF-α、IFN-β细胞因子的分泌量最高,与空白对照差异极显著,但均低于脂多糖(LPS)的作用;而加入TLR2抗体后,SNP组巨噬细胞RAW 264.7的上述各细胞因子的分泌量相较未加TLR2抗体组均降低,但除IL-6细胞因子外差异均不显著。由此可见,SNP可促进巨噬细胞RAW 264.7的增殖及细胞因子的分泌,但不是通过TLR2受体介导。

Fungus polysaccharides mainly mediate phagocyte secreting cytokines via Toll-Like Receptors(TLRs)to play a role in immunoregulation.To explore the effect of Sparassis latifolia neutral polysaccharides(SNP)on the secretion of macrophage cytokines and whether TLR2 mediates this process,in this study,using traditional water extraction method,SNP were isolated and purified from Sparassis latifolia via HZ-830 macroporous resins,DEAE-52 and Sepharose CL-6B.The molecular weight of SNP was determined by high performance gel permeation chromatography(HP-GPC),and the composition and structure characterization of monosaccharides were analyzed by ion chromatography(IC)and Fourier transform infrared chromatography(FT-IR).Then MTT colorimetric assay was used to analyze the effect of SNP on the proliferation activity of macrophage RAW 264.7.By the function of the optimal concentration of SNP,the content of cytokines secreted by macrophage RAW 264.7 was detected through ELISA.Finally,phagocyte acted by TLR2 antibody was treated by SNP,whether TLR2mediated the process that SNP regulated secretion of cytokines of macrophages RAW 264.7 were analyzed.The results showed that the molecular weight of SNP was 3.2×10;u and was composed of arabinose,galactose,glucose,xylose and mannose with a molar ratio of 6∶12∶63∶10∶5,without furanose.SNP in a certain concentration range could significantly promote the proliferation of RAW 264.7.The optimal concentration of SNP was 250μg/mL and the secretion of NO,IL-6,TNF-α,IFN-βin RAW 264.7 was the highest at this concentration,which were extremely significantly different from that of blank control,but the function was lower than that of LPS.However,after adding TLR2 antibody,the secretion of the above cytokines in macrophages RAW 264.7 in SNP group was decreased compared with the groups without TLR2 antibody,but the difference was not significant except for IL-6 cytokines.In conclusion,SNP could promote the proliferation and cytokine secretion of macrophage RAW 264.7,but this process was not mediated by TLR2.