基于VEGFR1/PDGFR信号通路的黄芪、莪术配伍促肿瘤新生血管正常化的机制研究

The preliminary study results proved that compatibility of Astragalus mongholicus and Rhizoma curcumae can effectively inhibit tumor growth and metastasis by inhibiting angiogenesis to some extent. According to the forefront information and related study results, applicants hypothesize that they can improve tumor hypoxia and reduce tumor cell malignant potential by promoting tumor angiogenesis normalization, improving connection pericytes and endothelial cells, and recovering oxygen and nutrient supply to tumor tissue. To understand the mechanism of promoting tumor angiogenesis normalization of Astragalus mongholicus and Rhizoma curcumae, a Hepatocellular Carcinoma fluorescence in situ model of nude rat will be established, the pericyte markers NG2 and vascular endothelial cell marker CD34 co-expression will be detected by double immunofluorescence staining method, tumor vascular wall integrity degree will be observed by scanning electron microscopy from morphology, the treatment effect will be evaluated as the index of tumor vessel maturation, vascular endothelial cells of human hepatocellular carcinoma in nude mice (Mice TECs) will be sorted by immunomagnetic beads sorting method, expression of VEGFR1, VEGFR2 and PDGFR in Mice TECs surface will be detected, the effect of drug intervention on VEGFR1/PDGFR signaling pathway will be observed. If successfully done, on one hand, the significance of tumor angiogenesis normalization will be revealed, on the other hand, we will find what channels are involved in the regulation of tumor angiogenesis of Astragalus mongholicus and Rhizoma curcumae, which is helpful for explaining the mechanism of antitumor by compatibility of Astragalus mongholicus and Rhizoma curcumae.

肿瘤血管形态、结构高度异质，在肿瘤侵袭转移中发挥重要作用。前期研究表明，黄芪、莪术配伍，补气活血，祛瘀生新，可有效改善肿瘤乏氧，干预新生血管生成，减少肿瘤侵袭转移。结合前期工作，申请者提出：二者通过促肿瘤血管形态、结构正常化，疏通氧供通道，从而纠正肿瘤乏氧，降低肿瘤细胞的恶性潜能；通过改善周细胞与内皮细胞的连接，减少新生血管机械性渗漏，降低循环肿瘤细胞的数量，从而遏制肿瘤细胞的侵袭转移。为证实以上假说，本研究利用肝癌原位荧光动物模型，以周细胞的密度评估微血管完整度，检测肝癌裸鼠外周血GFP标记的CTCs数，验证其通过促肿瘤新生血管正常化，发挥抗肿瘤效应。接着，采用免疫磁珠分选、内皮细胞体外成管等实验方法，基于VEGFR1/PDGFR信号通路对其作用机制进行深入研究。项目不仅可阐明黄芪莪术配伍促肿瘤新生血管正常化的分子机制，而且还可丰富和发展中药药性配伍理论，为临床安全合理用药提供科学依据。

本项目采用肝癌原位荧光动物模型，在活体成像系统下对肝癌裸鼠进行整体荧光影像观察，IF法检测肿瘤组织中NG2和CD34共表达，IHC法检测药物对肿瘤组织中HGF和c-met表达的影响，SEM观察肿瘤细胞形态及结构；体外分选裸鼠肝癌血管内皮细胞（Mice TECs），CCK-8检测细胞增殖，流式细胞术检测Mice TECs表面 VEGFR1、VEGFR2、PDGFR表达情况，免疫组化检测NG2、CD34与HIF-1α的表达，采用内皮细胞体外成管实验，体外检测EOMA、Mice TECs成管，比较体外加黄芪、莪术对EOMA、Mice TECs成管的差异，应用 VEGFR1/PDGFR选择性抑制剂Nintednaib，观察黄芪、莪术介导的成管的影响，深入探究黄芪、莪术配伍促肿瘤新生血管正常化的作用靶点及分子机制。.本项目结果验证了黄芪、莪术配伍是通过干预VEGFR1/PDGFR信号通路达到促肿瘤新生血管正常化而发挥抗肿瘤效应，黄芪+莪术（1:1配比）高剂量组平均瘤重及平均瘤体积均有明显降低，能显著降低肿瘤组织中HGF和c-met表达，肿瘤细胞出现明显的细胞膜破裂，细胞器溶解破裂，细胞核固缩；黄芪+莪术（1:1配比）高剂量组能明显抑制EOMA、Mice TECs细胞增殖，能显著增加EOMA、Mice TECs细胞CD34的表达，显著降低EOMA、Mice TECs细胞HIF-1α的表达。黄芪、莪术及黄芪+莪术对EOMA及MTECs细胞的血管生成有明显的改善作用，其增强EOMA及MTECs细胞成管与上调VEGFR1/PDGFR信号通路密切相关。.本项目初步探索了黄芪、莪术配伍对肝癌新生血管形态、结构、功能的影响，以及促肿瘤新生血管正常化的作用靶点及信号通路，从而解释二药配伍抗肿瘤的作用机制，为临床黄芪、莪术配伍使用治疗肝癌提供科学依据，为其应用提供一定参考。