中国科学院海洋研究所机构知识库

多肋藻(Costaria costata)属于多肋藻属，是一年生大型褐藻，主要分布于太平洋北部沿岸、日本北海道及大彼得湾等海域，具有悠久的食用历史。多年来，对多肋藻的研究大多集中在生态研究及人工养殖方面，对于其多糖的研究还不够深入，需要进一步研究。本文以多肋藻为研究材料，研究了多肋藻褐藻多糖硫酸酯的提取、分级纯化、降解等制备工艺。对获得的多肋藻褐藻多糖硫酸酯的理化性质和结构进行解析，同时对其生物活性进行系统研究，为多肋藻的全面开发利用奠定基础。

本研究从多肋藻中获得了多肋藻总糖CCP，通过分级纯化获得了F3、F4、F5三个多肋藻多糖分级组分。分析了CCP、F3、F4和F5的理化性质，并对 F5的多糖结构进行了解析，并通过体外、体内模型分别评价了CCP、F3、F4及F5的抗肺纤维化活性、抗肿瘤活性及免疫调节活性。主要实验结果如下：

1.   用热水浸提的方法从多肋藻中提取了多肋藻总糖CCP。CCP经过DEAE-Sepharose Fast Flow凝胶柱洗脱获得三种分级组分F3、F4及F5。化学组分分析表明CCP是一种以半乳糖、岩藻糖为主，硫酸基含量为18.54%，分子量为149846 Da的复杂多糖。F3是一种各种单糖含量比较平均的、硫酸基含量极低的杂多糖，重均分子量为57264 Da；F4是以岩藻糖为主、甘露糖次之的硫酸化岩藻聚糖，重均分子量为74005 Da；F5是以半乳糖和岩藻糖为主、其他单糖含量极低的硫酸化半乳岩藻聚糖，其重均分子量为75859 Da。

2.   通过核磁共振、红外光谱、质谱手段对F5进行结构解析。谱图分析结果表明，F5中主要有8种异头碳存在，多糖构型为α型；F5主要包含三种类型的多糖片段，分别是酸性岩藻寡糖、酸性半乳寡糖及酸性岩藻-半乳糖；寡糖聚合度为1~9，且含有不同个数的硫酸集团取代，硫酸基的取代主要发生在C-2、C-3位，在C-4位无硫酸基取代。

3.   通过使用小鼠肺纤维化模型对CCP的抗纤维化活性进行体内探究，发现CCP具有体内抗肺纤维化的活性。CCP治疗能够降低肺纤维化小鼠的炎症因子表达，抑制胶原蛋白沉积。进一步的研究发现，CCP可以通过降低COL2A1和p-Smad2/3的表达来抵抗TGF-β1诱导的上皮-间充质转化。

4.   对多肋藻总糖及其分级组分的抗肿瘤活性和免疫调节活性研究中发现，F3、F4、F5、CCP均能抑制肺癌细胞、胰腺癌细胞的生长，并促进A549、PANC-1细胞的细胞凋亡。在免疫调节活性评价中发现，低浓度下F4、F5及CCP不具有促炎活性，在高浓度下F4、F5及CCP均具有一定的促炎活性。F3在低浓度或高浓度下均具有抗炎活性；其余组分不具有抗炎活性。

综上实验，多肋藻褐藻多糖硫酸酯具有抗肺纤维化、抗肺肿瘤及免疫调节等生物活性，在生物医药领域具有很好的应用潜力。

Costaria costata belongs to Costaria Greville, is a large annual brown algae with a long history of consumption, mainly in the northern Pacific coast, Hokkaido, Japan and the Great Peter Bay. For many years, most of the research on C. costata has been focused on ecological studies and artificial culture, but the study of its polysaccharides is still not comprehensive enough and needs further research. In this study, the extraction, graded purification and degradation of fucoidan from C. costata (CCP) were investigated. The chemical properties and structure of CCP were analysed, and the biological activity was also systematically investigated to lay the foundation for the comprehensive exploitation of C. costata.

In this study, the CCP was isolated from C. costata, and three main graded fractions of CCP were obtained by graded purification named F3, F4, F5. The chemical properties of CCP, F3, F4 and F5 were investigated, and the structure of F5 was analysed. The anti-pulmonary fibrosis activity, anti-tumour activity and immunomodulatory activity of CCP, F3, F4 and F5 were evaluated by in vitro and in vivo models, respectively. The main experimental results are as follows:

1. CCP was extracted by hot water extraction and further eluted on a DEAE-Sepharose Fast Flow gel column to obtain three graded fractions F3, F4 and F5. Chemical composition analysis showed that CCP is a complex polysaccharide with galactose and fucose as the main components, with a sulfate group content of 18.54% and a molecular weight of 149846 Da. F3 is a heterogeneous polysaccharide with an average content of various monosaccharides and a very low content of sulfate group, with a molecular weight of 57264 Da; F4 is a sulfated fucose with a molecular weight of 74005 Da, mainly fucose, followed by mannose; F5 is a sulfated polysaccharide with a molecular weight of 75859 Da, mainly galactose and fucose, with a very low content of other monosaccharides.

2. The structural analysis of F5 was carried out by NMR, IR spectroscopy and mass spectrometry. The results of the spectral analysis showed that there were mainly eight kinds of heterocapitated carbons in F5, and the polysaccharide configuration was α-type; F5 mainly contained three types of polysaccharide fragments, separately, they are sulfated-fucose oligosaccharide, sulfated-galactose oligosaccharide and sulfated fucose-galactose oligosaccharide; the oligosaccharide polymerization degree was 1~9, and it contained different numbers of sulfate group substitutions, and the sulfate group substitutions mainly occurred at the C-2 and C-3 positions, and there was no sulfate group substitution at the C-4 position.

3. The anti-fibrotic activity of CCP was investigated in vivo using a mouse model of pulmonary fibrosis, and CCP was found to have in vivo anti-fibrotic activity. CCP treatment was able to reduce inflammatory factor expression and inhibit collagen deposition in mice with pulmonary fibrosis. Further studies revealed that CCP could resist TGF-β1-induced epithelial-mesenchymal transition by reducing the expression of COL2A1 and p-Smad2/3 in vitro.

4. In the study of the anti-tumor activity and immunomodulatory activity of CCP and its graded fractions, it was found that F3, F4, F5 and CCP could inhibit the growth of lung cancer cells and pancreatic cancer cells and promote apoptosis in A549 and PANC-1 cells. In the evaluation of immunomodulatory activity, it was found that F4, F5 and CCP did not have pro-inflammatory activity at low concentrations, while at high concentrations F4, F5 and CCP all had some pro-inflammatory activity. F3 had anti-inflammatory activity at both low and high concentrations; the remaining fractions did not have anti-inflammatory activity.

From the above experiments, we conclude that Fucose Sulfate of D. multifida has good potential for anti-pulmonary fibrosis, anti-pulmonary tumour, anti-pancreatic tumour and immunomodulatory applications.

第1章 绪论... 1

1.1 褐藻多糖硫酸酯... 1

1.1.1 褐藻多糖硫酸酯概述... 1

1.1.2 褐藻多糖硫酸酯的提取... 1

1.1.3 褐藻多糖硫酸酯的纯化... 3

1.2 褐藻多糖硫酸酯的结构研究... 4

1.2.1 褐藻多糖硫酸酯的结构研究手段... 4

1.2.2 褐藻多糖硫酸酯的结构研究现状... 4

1.3 褐藻多糖硫酸酯的活性研究... 5

1.3.1 抗肺纤维化活性... 5

1.3.2 抗肿瘤活性... 6

1.3.3 免疫调节活性... 6

1.3.4 其他生物活性... 7

1.4 多肋藻褐藻多糖硫酸酯... 7

1.4.1 多肋藻概述... 7

1.4.2 多肋藻褐藻多糖硫酸酯的结构研究进展... 7

1.4.3 多肋藻褐藻多糖硫酸酯的活性研究进展... 8

1.5 选题意义与研究内容... 9

第2章 多肋藻褐藻多糖硫酸酯的制备分离及结构解析... 11

2.1 引言... 11

2.2 实验材料和仪器... 11

2.2.1 实验材料... 11

2.2.2 实验试剂... 11

2.2.3 实验仪器... 12

2.3 实验方法... 13

2.3.1 多肋藻褐藻多糖硫酸酯的制备... 13

2.3.2 多肋藻褐藻多糖硫酸酯的分级纯化与降解... 13

2.3.3 理化性质分析... 14

2.3.4 光学与波谱学分析... 18

2.4 结果与讨论... 18

2.4.1 多肋藻褐藻多糖硫酸酯的制备... 18

2.4.2 多肋藻褐藻多糖硫酸酯的分级与纯化... 19

2.4.3 CCP及其分级组分的化学成分分析... 21

2.4.4 CCP及其分级组分的单糖组成分析... 23

2.4.5 CCP及其分级组分的红外波谱分析... 24

2.4.6 F5的质谱分析... 24

2.4.7 F5的核磁共振谱图分析... 29

2.5 小结... 32

第3章 CCP及其分级组分对肺纤维化的治疗作用评价... 35

3.1 引言... 35

3.2 实验材料和仪器... 35

3.2.1 实验材料... 35

3.2.2 实验试剂... 35

3.2.3 实验仪器... 36

3.3 实验方法... 36

3.3.1 CCP对小鼠肺纤维化的治疗作用研究... 36

3.3.2 CCP及分级组分抑制A549细胞EMT现象的研究... 39

3.4 结果与讨论... 41

3.4.1 CCP对小鼠肺纤维化的治疗作用... 41

3.4.2 CCP及分级组分抑制A549细胞EMT现象... 46

3.5 小结... 49

第4章 CCP系列组分的抗肿瘤活性及免疫调节活性评价... 51

4.1 引言... 51

4.2 实验材料和仪器... 51

4.2.1 实验材料... 51

4.2.2 实验试剂... 51

4.2.3 实验仪器... 51

4.3 实验方法... 52

4.3.1 抗肿瘤活性评价... 52

4.3.2 免疫调节活性评价... 52

4.4 结果与讨论... 54

4.4.1 CCP系列多糖的抗肺癌活性评价... 54

4.4.2 CCP系列多糖的抗胰腺癌活性评价... 55

4.4.3 CCP系列多糖的抗结肠癌活性评价... 56

4.4.4 CCP系列多糖的促炎活性评价... 57

4.4.5 CCP系列多糖的抗炎活性评价... 60

4.5 小结... 63

第5章 结论与创新点... 65

5.1 结论... 65

5.2 创新点... 65

参考文献... 68

致  谢... 78

作者简历及攻读学位期间发表的学术论文与其他相关学术成果... 80