基于“痰瘀阻络”理论探讨中药昆布活性成分岩藻黄素靶向调控Ran抑制胃癌淋巴管新生和转移的机制研究

Traditional Chinese medicine considers "phlegm-stasis blocking collateral" as the cause of gastric cancer. This study interprets the traditional Chinese medicine theory of " phlegm-stasis blocking collateral " from the perspective of lymphangiogenesis and metastasis of gastric cancer, and uses modern technology to elucidate the new mechanism of the anti-metastasis of gastric cancer by fucoxanthin from the traditional marine Chinese medicine kelp, and to analyze the target of fucoxanthin. Previous experiments have proved that fucoxanthin can inhibit the formation of tubular structures of human lymphatic endothelial cells induced by gastric cancer cell line (SGC7901), that may be related to the targeted regulation of Ran proteins. In this study, SGC7901 stable cell line without Ran function was constructed and the nude mouse lymph node metastasis model was established to elucidate the mechanism of fucoxanthin regulation on Ran. The fucoxanthin - fluorescence silicon nanoparticles were synthesized by chemical biological methods. Expression and purification of Ran protein, and the effect of fucoxanthin on the structure of Ran protein was detected by spectrophotometry. Amino acid point mutations were performed on Ran protein to analyze the structural interaction between fucoxanthin and Ran protein. This will enrich and improve the understanding of " phlegm-stasis blocking collateral ", also provide the basis and new ideas for the treatment of tumor lymph node metastasis, as well the research and development of new active ingredients of traditional marine Chinese medicine, which has important theoretical significance and clinical application value.

中医认为“痰瘀阻络”为胃癌的病因。本研究从胃癌淋巴管新生及转移这个视角解读中医传统理论“痰瘀阻络”，采用现代技术方法阐明海洋中药昆布活性成分岩藻黄素抗胃癌转移的新机制，解析作用靶点。课题组前期实验已证实岩藻黄素可抑制胃癌细胞系（SGC7901）诱导的人淋巴管内皮细胞管状结构形成，可能与其靶向调控Ran蛋白有关。本课题通过构建Ran功能缺失的SGC7901稳转细胞系，并构建裸鼠淋巴结转移模型，阐明岩藻黄素对Ran调控机制；采用化学生物学方法制备岩藻黄素-荧光硅纳米颗粒，验证其与Ran蛋白靶向结合作用；表达并纯化Ran蛋白，利用光谱技术检测岩藻黄素对Ran蛋白结构的影响；将Ran蛋白结合位点进行氨基酸点突变，解析岩藻黄素与Ran蛋白在结构上的相互作用。这将丰富和完善对“痰瘀阻络”的认识，并为新型海洋中药活性成分研发以及肿瘤淋巴结转移治疗提供依据和新思路，具有重要的理论意义和临床应用价值。

背景：淋巴结转移是胃癌转移的主要机制，淋巴管新生是淋巴结转移过程中的重要步骤。目前临床上尚无治疗胃癌淋巴结转移的药物。岩藻黄素是类胡萝卜素天然产物，是极具应用潜力的海洋藻类药源物质。关于岩藻黄素抑制肿瘤转移方面已有的研究成果主要集中在细胞外基质降解和血管转移方面，并且其在胃癌淋巴管新生和淋巴结转移中的作用未见报道。我们之前的研究表明岩藻黄素抑制淋巴管生成。然而，岩藻黄素的作用靶点和作用机制尚不清楚。.方法：采用细胞计数试剂盒-8 (CCK-8)和transwell实验评估岩藻黄素对细胞增殖和迁移侵袭的抑制作用。采用transwell小室将HGC-27细胞和HLECs细胞共培养，用于体外淋巴管生成的评价。采用组织芯片、生物信息学分析及分子对接分析了岩藻黄素在胃癌中可能的调控靶点。采用激光共聚焦、腺病毒转染及Real-Time qPCR和Western Blot等方法验证岩藻黄素调控通路。建立裸鼠足垫转移模型，采用免疫荧光技术检测VEGFR-3、LYVE-1和Ran蛋白在小鼠肿瘤组织中的表达及定位。.结果：组织芯片及生物信息学分析显示Ran蛋白在淋巴结转移组织中高表达，与预后不良相关。分子对接结果显示岩藻黄素与Ran的Met189和Lys167通过氢键相互作用。岩藻黄素通过下调Ran蛋白及importinβ表达，抑制NF-κB的核转运，从而抑制VEGF-C的分泌，在体内外抑制胃癌淋巴管生成和淋巴结转移。.结论：岩藻黄素通过调节Ran表达，并通过importinβ/NF-кB/VEGF-C核转运信号通路在体外和体内抑制胃癌诱导的淋巴管生成和转移。并为新型海洋中药活性成分研发以及肿瘤淋巴结转移治疗提供依据和新思路，具有重要的理论意义和临床应用价值。