類硫酸乙酰肝素多糖抗腫瘤和免疫調(diào)節(jié)作用及其機(jī)制研究
發(fā)布時(shí)間：2018-09-10 07:59
【摘要】：硫酸乙酰肝素(heparan sulfate,HS)是一類廣泛分布于動(dòng)物組織細(xì)胞表面和細(xì)胞外基質(zhì)中的線性多糖,其參與了眾多生理和病理過程,尤其在腫瘤的發(fā)生和發(fā)展中,HS介導(dǎo)腫瘤的增生、血管的形成、細(xì)胞的粘附和遷移、侵襲,甚至參與腫瘤微環(huán)境及機(jī)體的免疫調(diào)節(jié),為此以HS功能為靶點(diǎn)的探討和研究將為腫瘤干預(yù)提供新的思路。其中,HS類似物的制備和研究是較為常規(guī)的一種方式。大腸桿菌K5莢膜多糖(Escherichia coli K5 polysaccharide,K5PS)作為合成類硫酸乙酰肝素多糖的一種重要前體,其硫酸化衍生物在腫瘤發(fā)生和發(fā)展中的作用研究主要集中在K5PS硫酸化衍生物與FGF的相互作用及其介導(dǎo)的抗血管生成和抗腫瘤轉(zhuǎn)移的探討,而K5PS硫酸化衍生物與其他活性蛋白的相互作用及構(gòu)效關(guān)系研究尚未深入,其在間接抗腫瘤作用激活免疫方面的作用研究亦未見報(bào)導(dǎo)。為此,本研究將制備得到幾種不同硫酸化位點(diǎn)和程度的硫酸化衍生物,考察其直接抗腫瘤作用,即對腫瘤細(xì)胞增殖、粘附、遷移的影響;同時(shí)考察其間接抗腫瘤作用,即對免疫的調(diào)節(jié)作用;進(jìn)而進(jìn)一步分析硫酸化衍生物結(jié)構(gòu)與功能的關(guān)系,探討其抗腫瘤及免疫調(diào)節(jié)作用可能的機(jī)制和相關(guān)的信號通路。主要結(jié)果如下:(1)通過高密度生物發(fā)酵技術(shù)和離子交換色譜技術(shù)發(fā)酵、分離和純化了K5多糖,經(jīng)化學(xué)法硫酸化修飾后得到四種類硫酸乙酰肝素多糖:K5-OS_1、K5-OS_2、K5-NS和K5-NS,OS,分子量分別為65.81 KDa、68.86 KDa、9.32 KDa、10.19 KDa;硫酸化取代度分別為0.10、0.33、0.66、1.04。K5-OS_2的二糖結(jié)構(gòu)主要為△UA-GlcNAc6S和△UA2S-GlcNAc6S,即其主要發(fā)生6-O-硫酸化和2-O-硫酸化;而K5-NS,OS在N-硫酸化基礎(chǔ)上,再發(fā)生了6-O-硫酸化和2-O-硫酸化(△UA-GlcNS6S、△UA2S-GlcNS6S)。(2)制備所得的類硫酸乙酰肝素多糖中,K5-NS,OS具有明顯的抗腫瘤作用(抑制細(xì)胞增殖,抑制率為31.72%;抑制細(xì)胞粘附,抑制率為55.52%;抑制細(xì)胞遷移,抑制率為20.33%),K5-OS_2作用次之,K5PS、K5-OS_1和K5-NS未見明顯的作用。由此發(fā)現(xiàn),類硫酸乙酰肝素多糖結(jié)構(gòu)中硫酸化修飾位點(diǎn)和程度是決定其功能的重要因素,尤其是O位硫酸化基團(tuán),是介導(dǎo)其抗腫瘤作用的必要條件,N位和O位同時(shí)硫酸化可明顯增強(qiáng)其生物學(xué)活性。(3)K5-NS,OS能夠抑制CXCL12介導(dǎo)的黑色素瘤細(xì)胞的生長、粘附及轉(zhuǎn)移等生物學(xué)行為,進(jìn)一步研究發(fā)現(xiàn)其能夠抑制CXCL12介導(dǎo)的CXCR4的活化及其內(nèi)化,同時(shí)抑制下游Akt、ERK和JNK蛋白的磷酸化。(4)制備所得的類硫酸乙酰肝素多糖中,K5-OS_2具有最明顯的免疫增強(qiáng)活性(激活巨噬細(xì)胞和淋巴細(xì)胞),K5-OS_1和K5-NS,OS作用次之,K5PS和K5-NS未見明顯的作用。K5-OS_2刺激巨噬細(xì)胞后吞噬能力提高46.07%,NO釋放水平為249.23%(未刺激組設(shè)定為100%),TNF-α和IL-1β的分泌水平分別達(dá)到1180.47 pg/mL和123.17 pg/mL;K5-OS_2還能促進(jìn)淋巴細(xì)胞分泌細(xì)胞因子IL-2和IFN-γ和免疫球蛋白IgG1a和IgG2b。由此發(fā)現(xiàn),類硫酸乙酰肝素多糖結(jié)構(gòu)中,O位硫酸化基團(tuán)是介導(dǎo)其免疫增強(qiáng)活性的必要條件,而N位硫酸化對免疫調(diào)節(jié)活性影響不明顯。(5)K5-OS_2能夠激活巨噬細(xì)胞表面受體TLR4,隨后級聯(lián)激活下游MAPK和NF-κB信號通路,從而通過免疫增強(qiáng)作用達(dá)到間接抗腫瘤的目的。
[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.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：TQ281;R96
本文編號：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.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：TQ281;R96
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