类硫酸乙酰肝素多糖抗肿瘤和免疫调节作用及其机制研究
发布时间：2018-09-10 07:59
【摘要】：硫酸乙酰肝素(heparan sulfate,HS)是一类广泛分布于动物组织细胞表面和细胞外基质中的线性多糖,其参与了众多生理和病理过程,尤其在肿瘤的发生和发展中,HS介导肿瘤的增生、血管的形成、细胞的粘附和迁移、侵袭,甚至参与肿瘤微环境及机体的免疫调节,为此以HS功能为靶点的探讨和研究将为肿瘤干预提供新的思路。其中,HS类似物的制备和研究是较为常规的一种方式。大肠杆菌K5荚膜多糖(Escherichia coli K5 polysaccharide,K5PS)作为合成类硫酸乙酰肝素多糖的一种重要前体,其硫酸化衍生物在肿瘤发生和发展中的作用研究主要集中在K5PS硫酸化衍生物与FGF的相互作用及其介导的抗血管生成和抗肿瘤转移的探讨,而K5PS硫酸化衍生物与其他活性蛋白的相互作用及构效关系研究尚未深入,其在间接抗肿瘤作用激活免疫方面的作用研究亦未见报导。为此,本研究将制备得到几种不同硫酸化位点和程度的硫酸化衍生物,考察其直接抗肿瘤作用,即对肿瘤细胞增殖、粘附、迁移的影响;同时考察其间接抗肿瘤作用,即对免疫的调节作用;进而进一步分析硫酸化衍生物结构与功能的关系,探讨其抗肿瘤及免疫调节作用可能的机制和相关的信号通路。主要结果如下:(1)通过高密度生物发酵技术和离子交换色谱技术发酵、分离和纯化了K5多糖,经化学法硫酸化修饰后得到四种类硫酸乙酰肝素多糖:K5-OS_1、K5-OS_2、K5-NS和K5-NS,OS,分子量分别为65.81 KDa、68.86 KDa、9.32 KDa、10.19 KDa;硫酸化取代度分别为0.10、0.33、0.66、1.04。K5-OS_2的二糖结构主要为△UA-GlcNAc6S和△UA2S-GlcNAc6S,即其主要发生6-O-硫酸化和2-O-硫酸化;而K5-NS,OS在N-硫酸化基础上,再发生了6-O-硫酸化和2-O-硫酸化(△UA-GlcNS6S、△UA2S-GlcNS6S)。(2)制备所得的类硫酸乙酰肝素多糖中,K5-NS,OS具有明显的抗肿瘤作用(抑制细胞增殖,抑制率为31.72%;抑制细胞粘附,抑制率为55.52%;抑制细胞迁移,抑制率为20.33%),K5-OS_2作用次之,K5PS、K5-OS_1和K5-NS未见明显的作用。由此发现,类硫酸乙酰肝素多糖结构中硫酸化修饰位点和程度是决定其功能的重要因素,尤其是O位硫酸化基团,是介导其抗肿瘤作用的必要条件,N位和O位同时硫酸化可明显增强其生物学活性。(3)K5-NS,OS能够抑制CXCL12介导的黑色素瘤细胞的生长、粘附及转移等生物学行为,进一步研究发现其能够抑制CXCL12介导的CXCR4的活化及其内化,同时抑制下游Akt、ERK和JNK蛋白的磷酸化。(4)制备所得的类硫酸乙酰肝素多糖中,K5-OS_2具有最明显的免疫增强活性(激活巨噬细胞和淋巴细胞),K5-OS_1和K5-NS,OS作用次之,K5PS和K5-NS未见明显的作用。K5-OS_2刺激巨噬细胞后吞噬能力提高46.07%,NO释放水平为249.23%(未刺激组设定为100%),TNF-α和IL-1β的分泌水平分别达到1180.47 pg/mL和123.17 pg/mL;K5-OS_2还能促进淋巴细胞分泌细胞因子IL-2和IFN-γ和免疫球蛋白IgG1a和IgG2b。由此发现,类硫酸乙酰肝素多糖结构中,O位硫酸化基团是介导其免疫增强活性的必要条件,而N位硫酸化对免疫调节活性影响不明显。(5)K5-OS_2能够激活巨噬细胞表面受体TLR4,随后级联激活下游MAPK和NF-κB信号通路,从而通过免疫增强作用达到间接抗肿瘤的目的。
[Abstract]:Heparan sulfate (HS) is a kind of linear polysaccharides widely distributed on the cell surface and extracellular matrix of animal tissues. It participates in many physiological and pathological processes. Especially in the occurrence and development of tumors, HS mediates tumor proliferation, angiogenesis, cell adhesion and migration, invasion, and even participate in tumor microenvironment. Therefore, the study of HS function will provide new ideas for tumor intervention. The preparation and study of HS analogues is a more conventional method. Escherichia coli K5 polysaccharide (K5PS) is an important precursor to the synthesis of heparan sulfate-like polysaccharide. The interaction between K5PS sulfated derivatives and fibroblast growth factor and their mediated anti-angiogenesis and anti-tumor metastasis were mainly discussed. However, the interaction between K5PS sulfated derivatives and other active proteins and their structure-activity relationship were not yet well studied. The indirect anti-tumor effect of K5PS sulfated derivatives was investigated. In this study, we will prepare several sulfated derivatives with different sulfation sites and degrees to investigate their direct anti-tumor effects, that is, the effects on proliferation, adhesion and migration of tumor cells, and the indirect anti-tumor effects, that is, the regulation of immunity. The main results are as follows: (1) K5 polysaccharides were isolated and purified by high-density bio-fermentation and ion-exchange chromatography. Four sulfur-like compounds were obtained by chemical sulfation modification. Heparan acetate polysaccharide: K5-OS_1, K5-OS_1, K5-OS_2, K5-NS and K5-OS, K5-NS, K5-NS, OS, molecular weight were 65.81 KDa, 68.86 KDa, 9.32 KDa, 10.32 KDa, 10.19 KDa; sulfsubstitutiondegree of 0.10, 0.10.33, 0.66, 0.66, 1.04.04.K5-OS_2, the main disasaccharistructure of 91UA-GlcNAc 6S and 91UA2S-Glc NAc 6S-NAc 6S, that is, the main occurrenceof 6-O-O-O-O-O-O-sulfand 2-O-O-O-O-O-O-O-sulfation On this basis, 6-O-sulfation and 2-O-sulfation occurred again (delta UA-GlcNS6S, Delta UA2S-GlcNS6S). (2) Among the heparan sulfate polysaccharides, K5-NS and OS had obvious anti-tumor effects (inhibition of cell proliferation, inhibition rate was 31.72%; inhibition of cell adhesion, inhibition rate was 55.52%; inhibition of cell migration, inhibition rate was 20.33%) and K5-OS 2 action. Secondly, K5PS, K5-OS_1 and K5-NS had no obvious effect. It was found that the sulfated sites and degree of heparan-like sulfate polysaccharides were important factors to determine their function, especially O-sulfated groups, which were necessary to mediate their anti-tumor effects. Simultaneous sulfation of N-site and O-site could significantly enhance their biological activities. - NS and OS can inhibit the growth, adhesion and metastasis of CXCL12-mediated melanoma cells. Further studies have shown that OS can inhibit the activation and internalization of CXCR 4 mediated by CXCL12, and inhibit the phosphorylation of Akt, ERK and JNK proteins in the downstream. 4. Among the heparan sulfate polysaccharides, K5-OS_2 has the most obvious effect. Immunopotentiatory activity (activation of macrophages and lymphocytes), K5-OS_1 and K5-NS, OS, K5PS and K5-NS had no significant effect. K5-OS_2 stimulated macrophages and increased phagocytosis by 46.07%, NO release by 249.23% (100% in the non-stimulated group), and the secretion levels of TNF-a and IL-1 beta reached 1180.47 pg/mL and 123.17 pg/mL, respectively. S_2 can also promote the secretion of cytokines IL-2, IFN-gamma and immunoglobulin IgG1a and IgG2b by lymphocytes. It is found that O-sulfated group is a necessary condition in the structure of heparan sulfate-like polysaccharides to mediate their immune enhancement activity, while N-sulfated group has no significant effect on the immunoregulatory activity. (5) K5-OS_2 can activate the surface of macrophages. Receptor TLR4 then cascades to activate downstream MAPK and NF-kappa B signaling pathways, thereby indirectly anti-tumor through immune enhancement.
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TQ281;R96
本文编号：2233846
[Abstract]:Heparan sulfate (HS) is a kind of linear polysaccharides widely distributed on the cell surface and extracellular matrix of animal tissues. It participates in many physiological and pathological processes. Especially in the occurrence and development of tumors, HS mediates tumor proliferation, angiogenesis, cell adhesion and migration, invasion, and even participate in tumor microenvironment. Therefore, the study of HS function will provide new ideas for tumor intervention. The preparation and study of HS analogues is a more conventional method. Escherichia coli K5 polysaccharide (K5PS) is an important precursor to the synthesis of heparan sulfate-like polysaccharide. The interaction between K5PS sulfated derivatives and fibroblast growth factor and their mediated anti-angiogenesis and anti-tumor metastasis were mainly discussed. However, the interaction between K5PS sulfated derivatives and other active proteins and their structure-activity relationship were not yet well studied. The indirect anti-tumor effect of K5PS sulfated derivatives was investigated. In this study, we will prepare several sulfated derivatives with different sulfation sites and degrees to investigate their direct anti-tumor effects, that is, the effects on proliferation, adhesion and migration of tumor cells, and the indirect anti-tumor effects, that is, the regulation of immunity. The main results are as follows: (1) K5 polysaccharides were isolated and purified by high-density bio-fermentation and ion-exchange chromatography. Four sulfur-like compounds were obtained by chemical sulfation modification. Heparan acetate polysaccharide: K5-OS_1, K5-OS_1, K5-OS_2, K5-NS and K5-OS, K5-NS, K5-NS, OS, molecular weight were 65.81 KDa, 68.86 KDa, 9.32 KDa, 10.32 KDa, 10.19 KDa; sulfsubstitutiondegree of 0.10, 0.10.33, 0.66, 0.66, 1.04.04.K5-OS_2, the main disasaccharistructure of 91UA-GlcNAc 6S and 91UA2S-Glc NAc 6S-NAc 6S, that is, the main occurrenceof 6-O-O-O-O-O-O-sulfand 2-O-O-O-O-O-O-O-sulfation On this basis, 6-O-sulfation and 2-O-sulfation occurred again (delta UA-GlcNS6S, Delta UA2S-GlcNS6S). (2) Among the heparan sulfate polysaccharides, K5-NS and OS had obvious anti-tumor effects (inhibition of cell proliferation, inhibition rate was 31.72%; inhibition of cell adhesion, inhibition rate was 55.52%; inhibition of cell migration, inhibition rate was 20.33%) and K5-OS 2 action. Secondly, K5PS, K5-OS_1 and K5-NS had no obvious effect. It was found that the sulfated sites and degree of heparan-like sulfate polysaccharides were important factors to determine their function, especially O-sulfated groups, which were necessary to mediate their anti-tumor effects. Simultaneous sulfation of N-site and O-site could significantly enhance their biological activities. - NS and OS can inhibit the growth, adhesion and metastasis of CXCL12-mediated melanoma cells. Further studies have shown that OS can inhibit the activation and internalization of CXCR 4 mediated by CXCL12, and inhibit the phosphorylation of Akt, ERK and JNK proteins in the downstream. 4. Among the heparan sulfate polysaccharides, K5-OS_2 has the most obvious effect. Immunopotentiatory activity (activation of macrophages and lymphocytes), K5-OS_1 and K5-NS, OS, K5PS and K5-NS had no significant effect. K5-OS_2 stimulated macrophages and increased phagocytosis by 46.07%, NO release by 249.23% (100% in the non-stimulated group), and the secretion levels of TNF-a and IL-1 beta reached 1180.47 pg/mL and 123.17 pg/mL, respectively. S_2 can also promote the secretion of cytokines IL-2, IFN-gamma and immunoglobulin IgG1a and IgG2b by lymphocytes. It is found that O-sulfated group is a necessary condition in the structure of heparan sulfate-like polysaccharides to mediate their immune enhancement activity, while N-sulfated group has no significant effect on the immunoregulatory activity. (5) K5-OS_2 can activate the surface of macrophages. Receptor TLR4 then cascades to activate downstream MAPK and NF-kappa B signaling pathways, thereby indirectly anti-tumor through immune enhancement.
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TQ281;R96
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