【作者】 李辉；

【导师】 王静凤；

【作者基本信息】 中国海洋大学 ， 水产品加工及贮藏工程， 2011， 硕士

【摘要】 海参自古以来被视为珍贵的滋补品,其体壁中含有多种生物活性物质,如海参多糖、海参皂苷、脂肪酸、多肽、海参神经节苷脂等。海参硫酸多糖是海参体壁的重要功效成分,占海参体壁干重的7%-10%,包括海参硫酸软骨素(Sea Cucumber Chondroitin Sufate, SC-CHS)和海参岩藻聚糖硫酸酯(Sea Cucumber Fucan,SC-FUC)。其中,SC-CHS是一种带有岩藻糖支链且高度硫酸酯化的多糖。本实验中,SC-CHS提取自美国肉参,采用红外光谱分析和核磁共振波谱分析对其结构进行研究;通过细胞和动物实验,探讨SC-CHS抑制肿瘤生长和转移的作用及其机制。主要研究结果如下:美国肉参SC-CHS主要由葡萄糖醛酸(GlcUA)、氨基半乳糖(GalN)和岩藻糖(Fuc)组成,GlcUA、GalN和Fuc的摩尔比为1:0.7:0.9,岩藻糖支链95.9%为2,4-SO4取代;以多种肿瘤细胞株为研究对象,MTT法筛选出SC-CHS的敏感细胞株是人高转移肺癌细胞95D;流式细胞术分析证明SC-CHS可以诱导细胞早期凋亡;RT-PCR法研究证实SC-CHS促进了抑癌基因p53和p21 mRNA的表达,抑制了周期蛋白cycling B1和cdc2 mRNA的表达;下调细胞存活基因bcl-xl mRNA表达,提高半胱氨酸蛋白酶caspase-3 mRNA表达,提示SC-CHS能抑制95D细胞增殖,诱导细胞凋亡和G2/M期周期阻滞是其主要作用机制采用细胞粘附、迁移、侵袭实验和RT-PCR实验,探讨了SC-CHS体外抑制肿瘤转移的作用。结果表明,SC-CHS显著降低了肿瘤细胞同基质、内皮细胞的粘附能力(P<0.05);显著降低了95D细胞的迁移和侵袭能力(P<0.05);抑制细胞基质金属蛋白酶MMP-1/2的表达,促进其相应的基质金属蛋白酶抑制剂TIMP-1/2 mRNA的表达,通过降低肿瘤细胞在细胞外基质的侵袭能力发挥抑制肿瘤转移的作用。通过小管形成实验和鸡胚尿囊膜血管新生实验,发现SC-CHS具有抑制血管生成的作用。RT-PCR和Western Blot法检测表明SC-CHS可以降低细胞中血管内皮生长因子VEGF mRNA和蛋白的表达,提示SC-CHS通过抑制VEGF的活性,达到抑制血管新生作用。通过建立小鼠Lewis肺癌自发肺转移模型,研究SC-CHS在体内抑制肿瘤生长和转移的作用。结果表明,SC-CHS可以显著抑制荷瘤小鼠肿瘤的生长,其平均抑制率为31.5%;可以显著减少肺表面转移灶结节数(P<0.01),平均抑制率为47.5%;显著提高血清中TNF-α和γ-IFN的含量(P<0.05);降低荷瘤小鼠肿瘤组织中HIF-1α、VEGF mRNA的表达(P<0.05)。提示SC-CHS能显著抑制肿瘤细胞在小鼠体内的生长和转移,通过下调转移相关因子HIF-1α、VEGF的表达,抑制肿瘤诱导血管新生。通过建立建立小鼠B16人工肺转移模型,研究SC-CHS抑制B16实验性肺转移的作用。结果发现,SC-CHS可以显著降低小鼠的肺转移灶数量,平均转移抑制率为62.66%,并且改善了血清中肿瘤恶化的生化指标,减轻了肺脏的纤维化。提示SC-CHS可能通过影响B16细胞的活力,降低其粘附和侵袭能力来达到抑制肺转移的作用。综上所述,本文从体内和体外系统地研究了岩藻糖基化海参硫酸软骨素抑制肿瘤生长和转移的作用,并对其机制进行了探讨,为更好的阐明海参硫酸软骨素抗肿瘤活性的构效关系提供依据,为海参硫酸软骨素的抗肿瘤药物开发提供实验依据。更多还原

【Abstract】 Sea cucumber, an important food and medicine resource, has lots of bioactive substances, such as sea cucumber polysaccharide, saponins, collagen proteins and cerebrosides. The sea cucumber polysaccharide is one of the most important bioactive substance, and weight about 7%-10% of the body wall of the sea cucumber. There are sea cucumber chondroitin sulfate (SC-CHS) and the sulfated fucan of the sea cucumber polysaccharide. In this study, sea cucumber chondroitin sulfate (SC-CHS) was isolated from the Isostichopus badionotus, and its antitumor and antimetastasis effect was studied in tumor cells and animal models. Results are shown as follows:The monosaccharide composition of the SC-CHS was similar to that of chondroitin sulfate, mainly with molar ratio 1:0.7:0.9 of glucuronic acid (GlcUA), galactosamine (GlaN) and fucose (Fuc). IR and NMR analysis of anomer hydrogen signals of sulfate fucose in SC-CHS shows that mainly 2, 4-SO4 disubstituted. It was proved that SC-CHS could significantly inhibit 95D cell proliferation most sensitively by MTT assay; induce 95D cell early apoptosis with flow cytometry; SC-CHS induced expression of the p53 tumor suppressor gene and the Cdk inhibitor p21; The anti-proliferative activity of SC-CHS was accompanied by inhibition of cycling B1and Cdc2 mRNA; Moreover, SC-CHS inhibited the expression of bcl-xl,and induced expression of caspase-3 clearly. The results showed that SC-CHS significantly inhibit 95D cell proliferation through apoptosis and G2/M arrest.The effects of SC-CHS on adhesion, the invasion and metastasis ability of 95D cells were investigated through cell-base adhesion assay, migration assay, invasion assay and RT-PCR assay. The results showed that the SC-CHS could decrease the adhesion rate in dose dependent manner and inhibit the cellular motility(P<0.05); inhibited invsion of 95D cells in dose despondently(P<0.01); inhibited the expression of MMP-2/9, and induce expression of TIMP-1/2. The results suggested that the SC-CHS could inhibit the metastasis of 95D cell with protecting ECM under degradation by MMPs. The chorioallntoic membrane assay and tube formation assay indicate that SC-CHS inhibit the generation of new blood vessels. And RT-PCR and Western Blot assay confirm that its antiangiogenic effect was that SC-CHS can inhibit the expression of VEGF.The Lewis lung carcinoma models of C57BL/6J mice were established and the results showed that the tumor growth was suppressed significantly in SC-CHS groups, the average inhibition rate was 31.5% compared with control group. The metastatic foci occurrence rate of SC-CHS group was lower than control group, and the difference were significant(P<0.05). SC-CHS could significantly down-regulated the expression levels of HIF-1αand VEGF mRNA of the tumor than control. And SC-CHS also could increase amounts of TNF-αandγ-IFN in serum. The results suggested SC-CHS has inhibitory effect on growth and metastasis of Lewis lung carcinoma cell in mice, and may be through inhibiting solid tumor angiogenesis and improve the well-being of the immunity of the mice.To observe the inhibitory effect of SC-CHS on lung metastasis of mouse melanoma cell line B16-F10. The results suggested that SC-CHS clearly suppressed lung metastasis by 65.26% in mice and could reduce the levels of serum saliva acid content andγ-GT energy clearly (P<0.01). Biochemical parameters such as hydroxyproline, hexosamines and uronic acid levels in the lung were also reduced clearly (P<0.01) in the SC-CHS treated animals. Therefore, the results indicate that SC-CHS can inhibit the metastasis of B16-F10 metastatic mouse melanoma cells and its mechanism is maybe that it can inhibit the B16-F10 cells adhering to the ECM and reduce the migration of B16-F10 cells.In this study, we systemically investigated the antitumor and antimetastasis effect of a fucosylated chondroitin sulfate isolated from Isostichopus badionotus, furthermore we do lots of work to do that contribute to the illustration of the mechanism of its effect. Our works provide the scientific approvement for the research of the bioactive function of the sea cucumber polysaccharide and good use in food and medicine.更多还原