中国科学院海洋研究所机构知识库

肾纤维化是多种慢性肾病发展的终末阶段，肾脏上皮细胞的上皮间充质转化（EMT）是诱使肾纤维化的主要诱因之一，所以抑制EMT可有效地减缓肾脏的纤维化进程。褐藻多糖硫酸酯是一种褐藻中提取出的硫酸化多糖，已经被证明对多种肾脏疾病具有治疗作用。褐藻多糖硫酸酯的分子量对其生物活性有很大影响，本实验探究不同分子量褐藻多糖硫酸酯的制备及其在TGF-β1诱导的小鼠肾小管上皮细胞（MTEC）模型中的抗EMT活性。

（1）使用氧化降解法降解褐藻多糖硫酸酯（FPS）制备了不同分子量的褐藻多糖硫酸酯，通过探究反应时间、反应温度和氧化剂浓度对降解产物分子量的影响，得到了合适的降解工艺参数；根据降解产物的分子量，选取了合适的制备工艺，并制备了三批降解产物。通过对三批产物进行化学分析，验证了该工艺的稳定性，并选取其中一批化学成分相似但相对分子量分布在3.3 KDa到49.3 KDa的褐藻多糖硫酸酯样品（LHX 1-9）进行了单糖含量测定与红外光谱分析，结果表明，LHX 1-9与FPS间各单糖比例基本相同，且红外吸收峰的峰型与位置也基本相同，表明本降解工艺可以保持褐藻多糖硫酸酯的糖链结构不变。

（2）采用6ng/ml的TGF-β1对MTEC细胞进行建模，细胞出现典型的间充质细胞特征。细胞形态使用倒置显微镜进行观察和记录，细胞活力测定使用CCK-8试剂盒，EMT标志蛋白的表达使用蛋白免疫印迹法和细胞免疫荧光法进行检测。结果表明，LHX 1 (Mw=3.3 KDa) 和 LHX 3-9 (Mw=6.6 KDa, 8.3 KDa, 11.3 KDa, 14.9 KDa, 25.2 KDa, 35.4 KDa, 49.3 KDa)可以减少MTEC细胞中纤维连接蛋白（Fn）或结缔组织生长因子（CTGF）的表达，维持MTEC细胞正常的上皮细胞形态，抑制TGF-β1诱导的EMT转化。褐藻多糖硫酸酯的相对分子量和其抗EMT活性之间的关系是非线性的。LHX 1(Mw=3.3 KDa), LHX 5 (Mw=11.3 KDa)和LHX 8 (Mw=35.4 KDa)可以同时下调Fn蛋白和CTGF蛋白的表达，表现出更优的抗EMT活性。

Renal fibrosis is the end stage of the development of many chronic kidney diseases. Epithelial-mesenchymal transition (EMT) of renal epithelial cells is one of the main causes of renal fibrosis. So inhibiting the EMT phenomenon can effectively slow down the fibrosis of the kidneys. Fucoidans are fucose-containing sulfated polysaccharides extracted from brown algae and this polysaccharides have been shown to have therapeutic effects on a variety of kidney diseases. The molecular weight of Fucoidan sulfate has a great influence on its biological activity. We have investigated the anti-EMT effect of different molecular weight (Mw) fucoidans on TGF-β1-induced mouse renal tubular epithelial cell (MTEC) mode.

(1) Oxidative degradation method was used to obtain fucoidans with different molecular weights and the effect of reaction time, reaction temperature and the concentration of oxidants on degradation molecular weight were investigated. Then appropriate process parameters were obtained. According to the molecular weighr of degradation products, two suitable preparation process were selected and three batches of degradation products were prepared. After chemical analysis of three independent batches of prepared samples, we performed chemical analysis on three batches of products to verify the stability of the process, and one batch of fucoidan sample (LHX 1-9) which chemical contents are similar but Mw ranging from 3.3 KDa to 49.3 KDa were selected for monosaccharide content determination and infrared spectrum analysis, the results showed that the ratio of each monosaccharide between LHX 1-9 and FPS was basically the same, and the peak shape and position of the infrared absorption peak are also basically the same, which indicated that this degradation process can well protect the active functional groups and sugar chain structure of Fucoidan.

(2) 6ng / ml TGF-β1 was used to model MTEC cells, and the cells showed typical characteristics of mesenchymal cells. Cell morphology was observed and recorded by an inverted microscope, Cell viability was detected by CCK-8, and EMT markers expression were detected by Western-bolt and Cell immunofluorescence assay. As a result, we found LHX 1 (Mw=3.3 KDa) and LHX 3-9 (Mw=6.6 KDa, 8.3 KDa, 11.3 KDa, 14.9 KDa, 25.2 KDa, 35.4 KDa, 49.3 KDa) could resist the TGF-β1-induced depithelial–mesenchymal transition (EMT) by decreased expression of Fn or CTGF, and maintained epithelial cell morphology in MTEC. However, the relationship between the Mw of fucoidans and their anti-EMT effect is not simply linear. Among the samples, LHX 1(Mw=3.3 KDa), LHX 5 (Mw=11.3 KDa) and LHX 8 (Mw=35.4 KDa) showed significant anti-EMT effects than others by de-regulated Fn and CTGF expression on MTEC cells.

目 录

第一章 引 言.......................................................................... 1

1.1 海洋生物资源与海洋药物............................. 1

1.2 褐藻多糖硫酸酯的研究概况........................... 2

1.2.1 褐藻多糖硫酸酯的提取与降解工艺..................................... 3

1.2.2 褐藻多糖硫酸酯的多种肾脏保护活性................................. 3

1.2.3 褐藻多糖硫酸酯的结构与活性............................................. 8

1.3 肾纤维化与上皮间充质转分化......................... 9

1.4 选题意义与研究背景................................ 10

第二章 低分子量褐藻多糖硫酸酯的制备及质量研究...... 13

2.1 仪器与材料........................................ 13

2.1.1 实验仪器............................................................................... 13

2.1.2 实验材料............................................................................... 13

2.1.3 实验试剂............................................................................... 14

2.2 实验方法.......................................... 15

2.2.1 单因素实验........................................................................... 15

2.2.2 降解工艺研究与优化........................................................... 16

2.2.3 生产工艺验证....................................................................... 17

2.2.4 化学分析方法....................................................................... 18

2.3 实验结果.......................................... 21

2.3.1 氧化降解法的实验指标对产物分子量的影响................... 21

2.3.2 降解工艺确定....................................................................... 22

2.3.3 化学成分分析....................................................................... 25

2.3.4 生产工艺验证结果............................................................... 25

2.4 讨论.............................................. 26

第三章 不同分子量褐藻多糖硫酸酯抑制MTEC细胞上皮间充质转分化的研究............................................................ 29

3.1 实验仪器与材料.................................... 29

3.1.1 实验仪器............................................................................... 29

3.1.2 实验材料............................................................................... 29

3.1.3 实验试剂............................................................................... 29

3.2 实验方法.......................................... 31

3.2.1 细胞毒性实验....................................................................... 31

3.2.2 TGF-β1有效浓度筛选.......................................................... 32

3.2.3 样品有效作用时间研究....................................................... 32

3.2.4 细胞免疫荧光实验............................................................... 32

3.2.5 Western Blot实验.................................................................. 33

3.3 实验结果.......................................... 36

3.3.1 细胞毒性实验....................................................................... 36

3.3.2 TGF-β1作用浓度的确定...................................................... 37

3.3.3. 样品作用不同时间后的细胞活力与细胞形态.................. 38

3.3.4 细胞免疫荧光结果............................................................... 41

3.3.5 蛋白免疫印迹结果............................................................... 43

3.4 讨论.............................................. 44

第四章 结论与创新点.......................................................... 47

4.1 结论.............................................. 47

4.2 创新点............................................ 47

参考文献................................................................................ 49

致 谢...................................................................................... 55

作者简历及攻读学位期间发表的学术论文与研究成果.... 57