【作者】 范玉莹；

【导师】 周义发；

【作者基本信息】 东北师范大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 人参(Panax ginseng C. A. Meyer)是著名的中药,吉林省是中国人参的主要产区。人参的有效成分包括多糖、皂苷、蛋白及其他一些小分子类物质。研究表明人参多糖具有多种药理活性,但由于分离纯化较困难,人参多糖结构与活性的关系以及多糖作用的机制研究较少。本论文以人参多糖中不同结构的果胶为研究对象,研究它们对细胞迁移的影响,以揭示多糖构效关系及作用机理。人参多糖主要包括中性的淀粉样葡聚糖和酸性的果胶类多糖(GP)。实验结果表明,人参果胶能特异性抑制L-929细胞的迁移,与对照组细胞相比,人参果胶对细胞迁移速度的最大抑制可达到60%。它的抑制作用不受血清的影响,说明人参果胶是血清依赖和血清非依赖两种迁移途径的抑制剂。为了研究人参多糖的构效关系,我们按照本实验室建立的方法将人参果胶进行了系统分级,得到9种多糖级分,其中包括4种富含聚半乳糖醛酸(HG)结构域的级分(WGPA-1-HG,WGPA-2-HG,WGPA-3-HG,WGPA-4-HG);2种既含有HG结构域又含有I型聚鼠李半乳糖醛酸(RG-I)结构域的级分(WGPA-3-RG,WGPA-4-RG);1种富含葡聚糖的级分(WGPA-N);2种富含阿拉伯半乳聚糖(AG)的级分(WGPA-1-RG,WGPA-2-RG)。我们研究了每一种级分对细胞迁移速度的影响。结果显示,富含HG结构域的果胶级分对细胞迁移速度具有明显的抑制作用,其作用强度的顺序为WGPA-1-HG < -2-HG < -3-HG < -4-HG。当浓度为0.015 mg/mL时,WGPA-3-HG对L-929细胞迁移速度的抑制作用就具有显著性。WGPA-3-RG和WGPA-4-RG对L-929细胞迁移速度的抑制作用要强于富含HG结构域的果胶级分,而含有微量HG和RG-I结构域的WGPA-N、WGPA-1-RG和-2-RG三种级分,对L-929细胞迁移速度的抑制作用极其微弱。因此,这些不同结构类型的人参果胶对细胞迁移速度的抑制活性与HG结构域的含量和RG-I结构域的含量是密切相关的。为了证实RG-I型果胶对细胞迁移的作用,我们按照本实验室建立的方法制备了2种RG-I型果胶:g-RGI-low(RG-I含量较低)和g-RGI-high(RG-I含量较高)。细胞迁移实验结果显示,RG-I型果胶对细胞迁移具有较强的抑制作用,且呈浓度依赖关系,即使浓度低于0.015 mg/mL时,也具有明显的抑制活性。进一步研究发现,RG-I型果胶和HG型果胶的混合物对细胞迁移的抑制作用要比每一种果胶单独作用更明显,说明二者具有协同效应。在此基础上,我们对RG-I型果胶和HG型果胶的作用机理进行了研究。结果表明,二者的作用都需要果胶对细胞进行预处理,预处理后细胞的形态、大小、粘着斑、细胞骨架以及信号分子的酪氨酸磷酸化都发生了改变。因此推测,人参RG-I型和HG型果胶在细胞迁移过程中不单纯作为封闭剂。进一步研究表明,RG-I型和HG型果胶能够减弱细胞的粘附能力和降低细胞的伸展速度,说明果胶对细胞迁移的抑制作用可能是通过减弱细胞粘附能力以及降低细胞伸展速度实现的。此外,我们还探讨了果胶在细胞膜上的受体分子。由于果胶能够与半乳凝集素-3(galectin-3)特异结合并影响多种细胞行为,我们研究了galectin-3是否参与了果胶对细胞迁移的抑制作用。结果表明人参RG-I型和HG型果胶对细胞迁移的影响并不是galectin-3介导的。细胞迁移在肿瘤细胞转移和发生过程中发挥着重要的作用,目前抑制细胞迁移已成为抗肿瘤研究新的着手点。因此,本论文的研究结果将为人参多糖抗肿瘤活性机理研究提供新的数据支持,为人参的药理应用及产品开发提供理论依据。更多还原

【Abstract】 Panax ginseng C. A. Meyer has a long history of use as an herbal medicine in Asian countries. There are numerous different compounds in ginseng, including ginseng polysaccharides, ginsenosides, ginseng peptides and other small molecules. It has been reported that ginseng polysaccharides have many bioactivities. It is so hard to separate and purify the ginseng polysaccharides that the structural features related to the effects and the underlying mechanisms are less known. Therefore, we tested the effect of different fractions from ginseng polysaccharides on cell migration in this paper, and demonstrated on structure-activities and mechanisms in detail.Ginseng polysaccharides are mainly composed of neutral polysaccharides (starch-like glucans) and acidic substances (ginseng pectin, GP). In our study, ginseng pectin was examined for its effect on cell migration. Ginseng pectin impaired the migration of L-929 cells and reduced their migration speed by up to 60% of control. Cell migration was reduced to the same extent in the presence or absence of serum, which suggested that ginseng pectin acted as a common inhibitor to both serum-dependent and serum-independent migration pathways.To demonstrate the structure-activities, ginseng pectin was fractionated according to the previous methods in our lab, and nine fractions were characterized, including four homogalacturonan (HG)-domain rich fractions (WGPA-1-HG, WGPA-2-HG, WGPA-3-HG, WGPA-4-HG); two containing both HG- and Rhamnogalacturonan I (RG-I)-domains fractions (WGPA-3-RG, WGPA-4-RG); one glucan rich fraction (WGPA-N); two arabinogalactan rich fractions (WGPA-1-RG, WGPA-2-RG). We tested the effect of each fraction on cell migration. The HG-domain rich pectins caused significant inhibition on cell migration. The order of the inhibitory effects is WGPA-1-HG < -2-HG < -3-HG < -4-HG. At 0.015 mg/mL, WGPA-3-HG significantly inhibited L-929 cell migration. WGPA-3-RG and -4-RG showed a slightly stronger inhibition than the HG-domain rich fractions. WGPA-N, WGPA-1-RG and -2-RG, containing no, trace or minor HG and RG-I domains, showed less effects on cell migration. The inhibitory effect of ginseng pectic polysaccharides was related to HG domain content and RG-I domain content.To confirm the inhibitory effect of RG-I domain on cell migration, we prepared two kinds of RG-I-rich pectins: g-RGI-low (low RG-I domain content) and g-RGI-high (high RG-I domain content). The datas on cell migration showed RG-I-rich pectins inhibited cell migration in dose-dependent manner. Their inhibitory effects became significant even at less than 0.015 mg/mL. Further studies revealed that combination of HG- and RG-I-rich pectins exerted stronger effects than either HG- or RG-I-rich pectin alone.On the basis of results above, we studied the mechanisms of RG-I and HG pectins on cell migration. Experiment datas showed that the effects of RG-I- and HG-rich pectins were dependent on pretreatment and the pretreatment caused alterations in cell morphologies such as cell size and shape, focal adhesion, the organization of actin filaments, and the tyrosine phosphorylation of signaling molecules, suggesting that HG and RG-I pectins did not act simply as blocking agents during cell migration. Further studies showed that RG-I- and HG-rich pectin treatment decreased cell adhesion and cell spreading on the substratum, indicating that RG-I- and HG-rich pectins might exert their effects on cell migration via decreasing cell adhesion and cell spreading. Besides, we explored the target molecules on cell membrane of RG-I- and HG-rich pectins. Due to the ability of galectin-3 to bind some pectins, and has been postulated to mediate the actions of pectins in various cellular processes, we investigated if galectin-3 participated in RG-I- and HG-rich pectin mediated migration inhibition. The results suggested that RG-I- and HG-rich pectins did not target galectin-3 for their effects on cell migration.Cell migration plays an important role in cancerometastasis and tumorigenesis, impairment of cell migration has been the new tool for inhibiting tumor. Therefore, these findings represent a significant contribution towards understanding the anti-tumor activities of ginseng polysaccharides and applying them to health food and medicine.更多还原