以选择素相关因子为核心的黄芪、莪术配伍抗肿瘤转移机制研究

Since the metastasis of tumors is a fully dynamic process, the academic community is frequently controversial about the use of blood-activating medicine, and it is believed that it will help spread the metastasis. Inspired by the theory of dissemination and settling down through the The Inner Canon of Huangdi , according to a large number of literatures and previous research results, it is believed that tumor metastasis is a process combination of motion and quiescence, but the process of recruiting platelets by circulating tumor cells and forming a tumor thrombus and adhering to blood vessel walls is more static. In view of the corresponding qi deficiency and blood stasis, using gene transcriptomics methods, gene knockout and digital PCR techniques, the liver metastasis of colon cancer and selective knockout mice were studied to reveal the effects of the combination of Huangqi and Ezhu about E-selectin, P-selectin, and related cytokines (FGF2、VEGF-A, TGF-β, and IL-8) and the mechanisms that affect EMT, E-cadherin, N-cadherin, Vimentin, Snail, and STYK1. For the precise understanding of the key pathogenesis of tumor metastasis, the anti-tumor metastasis of blood circulation and phlegm drugs is extensive, deep, and reasonable to provide a solid theoretical basis.

囿于肿瘤转移为完全“动态”过程认识，学界对活血药运用频有争议，认为有助扩散转移之虞。受《内经》传舍理论启示，根据大量文献及前期课题研究成果，申请者认为：肿瘤转移动静结合，而以循环肿瘤细胞（CTCs）募集血小板形成癌栓黏附血管内壁体现为“静态”的过程更为关键。针对相应的气虚血瘀病机,运用基因转录组学方法，基因敲除和数字PCR技术，以结肠癌（CT26）肝转移及选择素基因缺失小鼠模型为研究对象，揭示黄芪、莪术配伍调控选择素（E -selectin，P- selectin）、相关细胞因子（FGF2、VEGF-A、TGF-β及IL-8）及对上皮间质转化途径（EMT，E-cadherin、N-cadherin、Vimentin、Snail和STYK1）影响的机制，为肿瘤转移关键病机精准认识，活血化瘀药抗肿瘤广泛、合理运用提供坚实的理论依据。

课题组前期筛选补气活血代表药对黄芪-莪术并聚焦肿瘤转移核心环节——肿瘤血管新生和异构，探寻其用药规律及生物学机制，提出了适时、恰量、确药使用其配伍可针对血管正常化、改善肿瘤微环境发挥抗转移作用。在此基础上，本课题总结文献、名老中医经验及团队多年临床实证，认为肿瘤转移动静结合，而以循环肿瘤细胞募集血小板形成癌栓黏附血管内壁体现为“静态”的过程更为关键，故选择核心指标——选择素，运用转录组学、代谢组学、基因敲除等技术，以结肠癌原位移植及选择素基因缺失小鼠模型为研究对象，揭示了黄芪-莪术配伍调控选择素（E-selectin，P-selectin）及相关信号通路发挥抗肿瘤转移核心机制，为活血类药抗肿瘤广泛、合理运用提供有力支撑。.结果：①黄芪-莪术抑制原位移植瘤生长及转移并增强模型小鼠免疫力、调节免疫微环境稳态；可抑制P-选择素表达、减少肿瘤细胞与血小板黏附。②P-选择素敲除鼠具有更低的结肠癌原位移植易感性并减轻结肠癌引起的免疫及代谢异常，黄芪-莪术可降低其血清中IL-2、IL-4、IL-5及INF-γ水平，增加IL-10水平、增加其外周血中NK细胞及细胞毒性T细胞和Th细胞来增加P-选择素敲除鼠免疫杀伤力并改善其异常的代谢组。③E-选择素异常可导致肿瘤恶性进展，黄芪-莪术发挥抗肿瘤作用时需调控其功能。④黄芪-莪术在抑制肿瘤转移时其潜在下游信号SERPINB1，ECT2等具有显著差异性。⑤黄芪-莪术可影响结肠癌细胞黏附力、可抗凝血和影响血小板聚集活性。⑥P-选择素在结肠癌细胞过表达直接增加了肿瘤细胞黏附和迁移能力，且黄芪-莪术未能对其黏附和迁移产生影响。⑦黄芪-莪术影响Lewis A和Lewis X的表达及分布；可下调N-cadherin，vimentin，snail、上调E-cadherin的表达；可显著降低S-Lewisx、PSGL-1、Mucin-1、Integrin等表达。.黄芪-莪术配伍，对选择素及其受体参与并调控的肿瘤转移信号通路有较为明确的效果，在其配伍抗肿瘤转移作用中选择素占核心的地位，结果为确定补气活血与减少黏附、抑制转移的对应关系，为补气活血抗肿瘤转移提供依据，并丰富和发展了中药配伍理论。