实验海洋生物学重点实验室知识库

癌症是世界范围内的主要公共卫生问题之一，其中，肝癌被列为第三大高致死率癌症和第六大常见肿瘤，每年约有78.2万例死亡和84.1万例新发病例。在过去的几十年里，肝癌的治疗取得了显著的进展。然而，这些治疗的主要缺点在于有效化疗栓塞后的进展和消融或手术切除后的复发。与其他肿瘤相比，肝癌的预后仍然较差。更严重的是，肝癌不仅是一种对化疗高度耐药的肿瘤，而且最适用的肿瘤化疗方案也受到潜在肝病问题的严重制约。因此，迫切需要新的药物治疗。海洋微生物多糖尤其是深海细菌胞外多糖因其具有好的生物相容性，低毒以及容易获得等优势，为开发高效低毒的抗肿瘤新药提供了可能。本文主要围绕一种深海溶藻弧菌Vibrio alginolyticus 364胞外多糖EPS364展开性质分析、抗肿瘤活性机制以及生物合成过程研究。

首先，通过醇沉方法，从V. alginolyticus 364发酵液上清中获得粗多糖，再经过DEAE-Sepharose Fast Flow阴离子柱和Sephadex G-100 column凝胶层析柱纯化后得到胞外多糖EPS364。通过NanoPhotometer^®微量分光光度计测定的紫外吸收光谱鉴定EPS364不含核酸和蛋白等杂质；通过HPGPC色谱测定，确定EPS364相对分子量为14.8 kDa；通过PMP衍生化的方法，经HPLC测定确定EPS364的单糖组成的摩尔比为甘露糖：葡糖胺：葡萄糖醛酸：半乳糖胺：阿拉伯糖 = 5 : 9 : 3.4 : 0.8 : 1.5；通过红外光谱分析，观察到EPS364含有多糖特征吸收峰；通过扫描电镜观察，EPS364呈现多孔均一网状结构。通过能谱扫描分析，EPS364元素占比为：C占48%，O占37%，P占4%，N占 6%，Na 占6%, Ca占 2%，符合多糖元素组成特征。

其次，对EPS364的抗肿瘤作用机制进行了研究。通过MTT实验，检测到EPS364可以选择性抑制地肝癌细胞Bel-7402和Huh7.5的生长，EPS364展现出明显的抗肿瘤作用，且这种抗肿瘤作用展现出时间和剂量依赖效应。EPS364对肝癌细胞Bel-7402和Huh7.5两种细胞系的抗肿瘤作用的IC₅₀约为0.4-0.5 mg/mL，即27-33.8 nM（相对分子质量为14.8 kDa）。通过倒置显微镜和扫描电镜观察到，EPS364诱导肝癌Huh7.5细胞逐渐聚集成团，细胞丝状伪足消失，细胞粘附能力减弱。并通过Annexin V-FITC/PI双染实验，证实了EPS364可诱导Huh7.5细胞凋亡。通过蛋白质组学和qRT-PCR的分析，分析了EPS364抗肿瘤的分子机理。EPS364显著下调Huh7.5细胞的细胞粘附分子、ECM-受体相互作用分子和FGF19-FGFR4信号通路分子。这些代表性显著下调的分子主要包括：FGFR4、KLB、CTNNB1、ALCAM、ICAM1、AFP、CAV1、CAV2、CDH2和FGF19。经过一系列文献的分析和研究发现，CTNNB1可以作为中间信号传导分子调控FGF19-FGFR4信号通路（FGF19、FGFR4和KLB），且CTNNB1的下调也与CDH2、ALCAM和ICAM1下调密切相关。Cav-1和Cav-2受FGFR4和CDH2的调节，并且还与肿瘤的进展、侵袭和转移相关。AFP是肝癌细胞生长和凋亡的生物标志物，其表达降低也证明了EPS364的抗肿瘤作用。ALCAM和ICAM1是有效的抗肿瘤靶点，在肿瘤细胞粘附和肿瘤的侵袭、迁移和转移中起着至关重要的作用。此外，通过蛋白质互作分析软件String 11.0，观察到这些下调分子（FGF19、FGFR4、KLB、AFP、CTNNB1、ALCAM、CDH2、CAV1、CAV2和ICAM1）存在着显著相互作用。因此构建了EPS364抗肿瘤分子机理模型，EPS364通过靶向FGF19-FGFR4信号通路，调控细胞内下游基因的表达来调节细胞粘附和生长，从而发挥抗肿瘤作用。

最后，分析了EPS364多糖的合成相关基因，构建了V. alginolyticus 364的遗传操作体系。分析了EPS364的合成机制，初步确定了控制EPS364多糖产量的基因，为EPS364多糖的合成调控研究奠定了基础。

Liver cancer is listed as the third leading cause of cancer death and the sixth most common neoplasm, with approximately 782,000 deaths and 841,000 new cases each year. In the past decades, significant advances have been made in the treatment of liver cancer. However, the major shortcomings of these treatments are progression after effective chemoembolization and recurrence after ablation or resection. Compared with other tumors, the prognosis of liver cancer still remains inferior. Liver cancer is not only a highly chemotherapy-resistant tumor, but the applicability of most chemotherapy regimens is severely limited by the underlying liver disease. Therefore, new treatments are urgently needed. Marine microbial polysaccharides, especially the exopolysaccharides of deep-sea bacteria, have the advantages of good biocompatibility, low toxicity and easy access, which provide the possibility for the development of new anti-tumor drugs with high efficiency and low toxicity. This thesis mainly focused on the properties analysis, anti-tumor activity mechanism and biosynthesis process of a exopolysaccharide, EPS364, purified from deep-sea Vibrio alginolyticus 364.

Firstly, the crude polysaccharide was obtained from the fermentation broth of Vibrio alginolyticus 364 by alcohol precipitation method, and then purified by a DEAE-Sepharose Fast Flow anion column and Sephadex G-100 column gel chromatography column to obtain the extracellular polysaccharide EPS364. EPS364 is free of nucleic acid and protein according to ultraviolet absorption spectrum which was detected by Nanophotometer^® microspectrophotometer. The relative molecular weight of EPS364 was 14.8 kDa which was examined by HPGPC chromatography. By means of PMP derivation, HPLC detection showed that EPS364 consisted of mannose, glucosamine, gluconic acid, galactosamine and arabinose with a molar ratio of 5:9:3.4:0.5:0.8. The characteristic absorption peak of polysaccharide in EPS364 was observed by infrared spectrum analysis. By scanning electron microscopy, EPS364 showed a porous homogeneous network structure. Through energy spectrum scanning analysis, the six elements of EPS364 is C, O, P, N, Na and Ca with the proportions of 48%: 37%: 4%: 3%: 6%: 2%, which accords with the element composition characteristics of a polysaccharide.

Secondly, the anti-tumor mechanism of EPS364 was studied. Through the MTT experiment, it was detected that EPS364 selectively inhibited the growth of hepatocarcinoma cells Bel-7402 and Huh7.5. EPS364 exhibits a significant anti-tumor effect, and this anti-tumor effect exhibits a time- and dose-dependent effect. The IC50 of the anti-tumor effect of EPS364 on the two cell lines of liver cancer cells Bel-7402 and Huh7.5 is approximately 0.4-0.5 mg/mL, which is 27-33.8 nM (relative molecular mass is 14.8 kDa). It was observed by inverted microscope and scanning electron microscope that EPS364 induced hepatocarcinoma Huh7.5 cells to gradually aggregate into clusters, and the cell adhesion ability was weakened. The Annexin V-FITC/PI double staining experiment confirmed that EPS364 induced cell apoptosis. Through proteomics and qRT-PCR analysis, the anti-tumor molecular mechanism of EPS364 was analyzed. EPS364 significantly down-regulates cell adhesion molecules, ECM-receptor interaction molecules and FGF19-FGFR4 signaling pathway molecules in Huh7.5 cells. These representative and significantly down-regulated molecules mainly include: FGFR4, KLB, CTNNB1, ALCAM, ICAM1, AFP, CAV1, CAV2, CDH2 and FGF19. After a series of literature analysis and research, it was found that CTNNB1 was an intermediate signaling molecule to regulate the FGF19-FGFR4 signaling pathway (FGF19, FGFR4 and KLB), and the down-regulation of CTNNB1 is also closely related to the down-regulation of CDH2, ALCAM and ICAM1. Cav-1 and Cav-1 -2 is regulated by FGFR4 and CDH2, and also related to tumor progression, invasion and metastasis. AFP is a biomarker for the growth and apoptosis of liver cancer cells, and its reduced expression also proves the anti-tumor effect of EPS364. ALCAM and ICAM1 are effective anti-tumor targets, which play a vital role in tumor cell adhesion and tumor invasion, migration and metastasis. In addition, through the protein interaction analysis software String 11.0, significant interactions of these down-regulated molecules (FGF19, FGFR4, KLB, AFP, CTNNB1, ALCAM, CDH2, CAV1, CAV2, and ICAM1) were observed. Therefore, the EPS364 anti-tumor molecular mechanism model was constructed. EPS364 targets FGF19-FGFR4 signaling pathway, and then regulates the expression of downstream genes within the cell to adjust the cell adhesion and growth, thereby exerting antitumor effects

Finally, the genes related to the synthesis of EPS364 polysaccharide were analyzed, and the genetic manipulation system of Vibrio alginolyticus 364 was constructed. The synthesis mechanism of EPS364 was analyzed, and the gene controlling the polysaccharide titer of EPS364 was preliminarily identified, which laid a foundation for the study on the synthesis and regulation of polysaccharide EPS364.