IOCAS-IR
海带多糖酸解产物的分离分析及免疫调节作用研究
其他题名Separation, structural analysis and immunomodulatory activity of acidic degradation products of polysaccharides from Saccharina japonica
耿丽华
学位类型博士
导师张全斌
2018-05-13
学位授予单位中国科学院大学
学位授予地点中国科学院海洋研究所
学位名称理学博士
学位专业海洋生物学
关键词海带 酸性多糖 硫酸化岩藻寡糖 杂多糖 结构分析 质谱 免疫调节
摘要

   来源于海带的酸性多糖在结构和组成上均表现出复杂、不均一的特性。采用无机酸降解海带多糖是常用的多糖解聚方法,此外,醇沉也是传统的纯化海带多糖的方法。提取过程、分离纯化方法及降解流程的不同都会导致海带多糖的分子量、结构、组成上的差异,进而导致活性的差异。本文对海带多糖酸解后的产物进行了较为系统的研究,通过探究海带多糖酸解及沉降的条件,得到了多种结构和组成较特殊的组分,并对相应组分的活性进行了考察;从酸解产物中制备了系列硫酸化岩藻寡糖,并对主要寡糖进行了结构表征;探讨了具有强免疫调节作用的硫酸杂多糖的细胞水平的免疫调节机制。具体的有:

    1 通过设置降解时间、沉降浓度以及选择不同沉降剂,对酸解得到的产物的化学组成及单糖组成进行了分析,通过比较,总结不同条件对产物的影响。在此基础上设置不同的流程制备了7个组分(OF1OF2OF3OF4OF5PF1SHP),包括水解条件下断裂的寡糖组分和较难水解的多糖组分,其中寡糖组分OF1-OF5为单糖组成以岩藻糖为主的寡糖混合物,PF1SHP为杂多糖,分子量大于20 kD。活性筛选结果显示,寡糖组分均不能刺激小鼠腹腔巨噬细胞(RAW 264.7)分泌一氧化氮,而多糖组分表现出明显的刺激作用;在马兜铃酸致大鼠肾小管上皮细胞(NRK-52E)损伤的模型中,只有OF3OF50.5 mg/mL时,以及PF10.5 mg/mL1 mg/mL)表现出肾保护作用(p < 0.05),其余组分没有活性。

    2 结合薄层色谱、电喷雾质谱和离子交换层析等手段,对OF1PF1进行了化学分析及活性考察。OF1主要为硫酸化的岩藻寡糖片段,不能激活巨噬细胞,但是其离子交换层析的分级组分却具有明显的免疫诱导作用;在PF1分离组分中,发现一种葡糖岩藻半乳聚糖,其单糖组成中半乳糖:岩藻糖=2:1,具有显著的肾保护作用。

    3 采用凝胶过滤色谱技术对OF2进行了分离,制备了系列硫酸化岩藻寡糖共6个组分,采用串联质谱方法对其中的7种单硫酸化和二硫酸化岩藻寡糖进行了结构表征,包括单硫酸化岩藻单糖、二糖、三糖、四糖 (FucSFuc2SFuc3SFuc4S),二硫酸化岩藻三糖、四糖、五糖 (Fuc3S2Fuc4S2Fuc5S2)6个组分中,只有F1F2F3可以抑制LPS诱导巨噬细胞的一氧化氮分泌,其他三个组分没有活性。

   4 SHP为具有强免疫调节作用的低硫酸化杂多糖,经降解后发现其含有的结构单元中,以葡萄糖醛酸寡糖(聚合度1-11)和甘露葡萄糖醛酸寡糖为主,还有少量的硫酸岩藻寡糖、杂合岩藻糖-甘露糖-葡萄糖醛酸寡糖。对SHP细胞水平的免疫诱导机制进行了研究,发现SHP作用于RAW 264.7巨噬细胞后,它可以被细胞表面Toll样受体-4识别,并产生相应信号,之后激活蛋白激酶B和丝裂原活化蛋白激酶,从而将信号传输到细胞核内,进而激活核因子κB和激活子蛋白与炎症相关基因结合,从而引发炎症因子的转录和表达。

其他摘要

     The sulfated polysaccharides derived from Saccharina japonica exhibit complex and inhomogeneous characteristics both in structure and composition. Degradation of polysaccharide by inorganic acid is a commonly used depolymerization method for polysaccharide. In addition, alcohol precipitation is a traditional method of purifying water-soluble polysaccharide. Different extraction processes, separation and purification methods and degradation processes lead to differences in the molecular weight, structure, and composition of the polysaccharides, which in turn lead to differences in bioactivity. This article systematically studied the acidic degradetion products of polysaccharide from Saccharina japonica. Through the study of different preparation processes of oligosaccharides and polysaccharides, different fractions with variety of structures and compositions were obtained, and the activities of the corresponding components were investigated. A series of sulfated fucooligosaccharides were prepared from low molecular weight components, and the structure of the main oligosaccharides were characterized. The mechanism of the immunomodulatory activity of the sulfated hetropolysaccharide was investigated. Specifically, that are:

      1 By setting degradation time, precipitation concentration and choosing different settling agents, the chemical composition and monosaccharide composition of the products obtained by acid hydrolysis were analyzed. Through comparison, the influence of different conditions on the products was summarized. Based on this, different processes were set up to prepare seven components, including both pieces of oligosaccharides and components difficult to hydrolyze, named OF1, OF2, OF3, OF4, OF5, PF1 and SHP, respectively. OF1-OF5 are oligosaccharides mainly composed of fucose, while PF1 and SHP are heteropolysaccharides with a molecular weight no lower than 20 kD. The activity screening results showed that low molecular weight fractions could not stimulate the secretion of nitric oxide from RAW 264.7 macrophages, while the large molecular weight fraction showed significant stimulatory effect. The damage of NRK-52E cells induced by aristolochic acid was constructed. In the cell model, only OF3 and OF5 at 0.5 mg/mL and PF1 0.5 mg/mL and 1 mg/mL (p < 0.05) showed a statistically significant protective effect on renal cell injury, and none of the other components showed activity.

       2 To elucidate the structure of OF1 and PF1, several separation methods combined with electrospray ionization mass spectrometry were employed, including preparative thin layer chromatography, strong anion exchange chromatography. OF1 mainly composed of sulfated fucose oligosaccharide fragments, and other heterosaccharides. The previous results showed that OF1 could not activate macrophages, but its fractionation components separated by ion chromatography had a significant immune induction. While a glucofucogalactan (F2) was obtained from PF1 by membrane filtration, which had a significant renal protective effect.

       3 The OF2 was separated by gel filtration chromatography. Six components of sulfated fucose oligosaccharides (named F1-F6) were prepared. Tandem mass spectrometry method for the 7 kinds of monosulfated and disulfated fucosaccharides has carried on the structure characterization, including monosulfated fucose (FucS), monosulfated fucobiose (Fuc2S), monosulfated fucotriose (Fuc3S), monosulfated fucoterose (Fuc4S), disulfated fucotriose (Fuc3S2), disulfated fucoterose (Fuc4S2), disulfated fucopentaose (Fuc5S2). The anti-inflammatory effects of the six components were examined and the results showed that only F1, F2, and F3 could inhibit the release of nitric oxide from LPS-induced macrophages, and the other three components had no activity.

     4 SHP is a low-sulfated heteropolysaccharide. After degradation, it was found that its structural units contained glucuronan oligosaccharides (degree of polymerization 1-11) and mannoglucuronan oligosaccharides, and a small amount of sulfated fucosaccharide, heterozygous fucose-mannose-glucuronan oligosaccharides. Studies have been conducted on the immunomodulatory mechanism of SHP in vitro, and it was found that after SHP acted on RAW 264.7 macrophages, it can be recognized by the cell surface TLR-4 and generated the corresponding signal, which activated protein kinase B and mitogen-activated protein kinase, then transmited signals to the nucleus, activated the binding of nuclear factor kappa B and activator proteins to inflammation-related genes, thereby triggering the transcription and expression of inflammatory factors.

语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/154526
专题中国科学院海洋研究所
第一作者单位中国科学院海洋研究所
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耿丽华. 海带多糖酸解产物的分离分析及免疫调节作用研究[D]. 中国科学院海洋研究所. 中国科学院大学,2018.
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