以趋化因子及其受体为中心研究黄芪莪术配伍对肿瘤血道转移、淋巴转移及免疫的综合调控机制

Preliminary research suggests that Huangqi Ezhu are compatible with the regulation of VEGF and the decrease of miRNA expression in ovarian cancer, liver cancer angiogenesis, vascular network reconfiguration, increase the sensitivity of chemotherapy drugs, the effect is remarkable. Subject to the overall concept of traditional Chinese medicine and uphold the Enlightenment of the theory, this topic proposed regulation of blood metastasis, lymphatic metastasis and improve the immunity of the synergy of the three importance to control tumor growth and metastasis. And pioneering the introduction of chemokines and their receptors as a central index communicated with the first three. Transfer model through the establishment of rat colon rectal injection, observation of chemokines and chemokine receptors (CXCL12/CXCR4, CXCL10/CXCR3, CCL21/CCR7, CCL3/CCR5) effect on tumor angiogenesis, lymph node metastasis and immune of the three, and to observe the intervention effect of the compatibility of Radix Astragali Rhizoma curcumae. In order to clarify the compatibility of Huangqi Ezhu "righting" enhance immunity, "Quxie" molecular mechanism of inhibition of angiogenesis, lymph node metastasis, it is important to the establishment of Chinese multi targets, multiway for the treatment of tumor after comprehensive efficiency advantage.

前期课题研究显示黄芪莪术相须配伍通过调控VEGF、miRNA的表达减少卵巢癌、肝癌新生血管生成，血管网重构，增加化疗药物的敏感性，效果显著。受中医整体观念及扶正祛邪理论的启示，本课题提出调控血道转移、淋巴道转移及提高机体免疫三者协同作用对控制肿瘤生长转移的重要性。并开创性地引入趋化因子及其受体作为连通三者的中枢指标。通过建立大鼠大肠癌直肠注射转移模型，观测趋化因子及受体（CXCL12/CXCR4、CXCL10/CXCR3、CCL21/CCR7、CCL3/CCR5）对肿瘤血管生成、淋巴转移及免疫三者的影响，同时观察黄芪莪术配伍的干预效应。以期阐明黄芪莪术配伍“扶正”增强免疫，“祛邪”抑制血管生成、淋巴转移的分子机制，这对确立中药多耙点、多途径治疗肿瘤综合取效的优势具有重要意义。

目的：以趋化因子及受体为中心研究补气活血药对黄芪、莪术配伍以及联合化疗药对结肠癌模型肝血道转移、淋巴转移及免疫功能的作用及机制。方法：一、体外CCK-8法测定黄芪莪术对结肠癌细胞活力的影响；Transwell法测定黄芪莪术对细胞迁移侵袭能力的影响；免疫荧光、Western Blot和RT PCR法检测黄芪莪术对肝转移相关信号通路关键蛋白的影响；过表达CXCR4、Wnt/β-catenin通路激动剂验证黄芪莪术抑制肝转移与CXCR4、Wnt/β-catenin通路和EMT之间的关系。二、动物实验构建小鼠结肠癌CT26.WT原位移植瘤模型，观察补气活血药黄芪、莪术配伍以及联合化疗药（顺铂/奥沙利铂/5-氟尿嘧啶）对模型小鼠原位移植瘤生长、转移及免疫功能的影响；Western Blot和RT PCR法检测CXCL12、CXCR4、β-catenin蛋白和mRNA表达；检测肿瘤转移及免疫功能相关因子CXCR3/CCR6/CXCL9/CCL20蛋白和mRNA表达；免疫组化法D2-40标记计数微淋巴管密度（MLVD），Western Blot检测淋巴转移相关因子VEGFC、VEGFD和CCL21/CCL19/CCR7蛋白表达；流式细胞术检测免疫相关趋化因子及受体CXCL10/CXCR3、CCL3/CCR5的表达。结果：一、黄芪莪术浓度依赖性降低结肠癌细胞的活力，抑制其肝转移潜能；降低CXCR4、Wnt/β-catenin通路和EMT通路关键蛋白表达，过表达CXCR4、Wnt/β-catenin信号通路激动剂验证了这一结果。二、动物实验黄芪、莪术配伍对原位瘤的生长和转移具有抑制作用，平均瘤重及转移率均低于模型组，黄芪莪术能够提高化疗药的敏感性；与模型组相比，黄芪莪术配伍特别是联合化疗药组，能够减少MLVD密度，降低CXCR3/CCR6蛋白和mRNA表达，降低VEGFC、VEGFD和CCL21/CCL19/CCR7蛋白表达，降低CXCL10/CXCR3蛋白和mRNA表达（P＜0.05或P＜0.01）。结论：黄芪、莪术配伍，能够抑制结肠癌细胞活力和侵袭转移能力，趋化因子及其受体参与并调控肝转移信号通路，联合化疗药后抑制原位瘤生长转移，提高化疗药敏感性，显著降低与肿瘤转移及免疫相关趋化因子及受体的表达，结果为补气活血药抑制结肠癌肝血道、淋巴转移，增强免疫提供了实验依据。