基于EMT相关转录因子的汉黄芩苷促肿瘤血管正常化作用及机制研究

Tumor angiogenesis is the basis of tumor growth and metastasis. Currently, the treatment of tumor blood vessels has received tremendous attention. The existing anti-angiogenic drugs mainly target VEGF/VEGFR pathway, which may damage blood vessels seriously and induce the activation of other pro-angiogenesis factors compensatorily in the tumor microenvironment, causing drug resistance and recurrence. The normalization of tumor blood vessels is a new concept to inhibit tumor angiogenesis and improve their function. Previous studies have shown that wogonoside promotes blood vessel normalization through inhibiting angiogenesis, reducing vascular permeability and improving vascular function. Mechanically, it relates to the intervention of EMT related transcriptional factors. However, the relationship between EMT related transcription factors and tumor vascular normalization, as well as the exact mechanisms of the improvement of wogonoside to tumor vessels remains unknown. This project aims to establish co-culture systems, orthotopic implantation and other in vivo and in vitro models as well as 3D live cell monitoring techniques to clarify and confirm the effect of wogonoside to tumor vascular normalization, explore the effect of EMT related transcriptional factors, especially their signal pathways and elaborate the mechanisms of wogonoside. Moreover, we aim to set up an anticancer drug screening platform for laying the foundation of vascular normalization treatment.

肿瘤血管是肿瘤赖以生长和转移的基础，针对肿瘤血管的治疗是目前研究热点之一。现有抗肿瘤血管药物主要针对VEGF/VEGFR通路，长期使用严重破坏血管，并诱发微环境中其他因子代偿性激活，引起肿瘤耐药及复发。肿瘤血管正常化是对抑制肿瘤新生血管并改善其功能的全新阐述。本课题前期研究表明，汉黄芩苷通过抑制肿瘤血管生成、降低血管通透性、改善血管功能从而促进肿瘤血管正常化，达到有效治疗肿瘤的目的，且其机制与干预EMT相关转录因子有关。但是EMT相关转录因子与肿瘤血管正常化的关系，汉黄芩苷改造血管的确切作用机制国内外文献未见报道。本项目旨在建立共培养体系、原位接种等体内外模型，利用3D活细胞监测等技术阐明并确证汉黄芩苷促进血管正常化的作用，探索EMT相关转录因子对肿瘤血管正常化的影响，重点围绕EMT相关转录因子的调控信号，研究汉黄芩苷的作用机制，并建立此类抗肿瘤药物的筛选平台，为促血管正常化治疗奠定基础。

血管正常化是当前血管异常性疾病的研究热点，由于血管正常化能够降低原位肿瘤转移能力、改善肿瘤微环境、增强肿瘤免疫效应，因此抗肿瘤血管治疗在抑制新生血管的同时也需要修复肿瘤血管，维持正常功能。本项目通过建立人白血病细胞U937移植瘤模型和人乳腺癌细胞MDA-231原位移植瘤模型，分别模拟血液瘤的骨髓微环境和实体瘤的原发灶微环境，发现了汉黄芩苷可以通过抑制血管生成、促进血管正常化，减缓肿瘤生长、抑制肿瘤转移、延长小鼠生存期。接着，通过在体外建立多种细胞的共培养模型，利用Transwell实验、小管形成实验、鸡胚尿囊膜实验和Matrigel Plug实验等进一步确证了汉黄芩苷能够抑制肿瘤微环境诱导的内皮细胞激活和增殖。Elisa实验也显示汉黄芩苷主要是降低了共培养体系中IL-8、MIP-1α和TNF-α的表达、分泌，从而抑制血管生成、促进正常化的。另一方面，本项目还发现汉黄芩苷可以通过调控细胞内的JAK-STAT3、NF-κB等信号通路，抑制肿瘤细胞和内皮细胞中EMT相关转录因子Twist1的表达，从而降低了细胞分泌基质金属蛋白酶重构细胞外基质的能力。此外，本项目还发现了其他结构类似的千层纸素苷和LYG-202也具有抑制血管生成、促进血管正常化的作用，深化了天然产物在调控复杂血管微环境中的独特作用。综上所述，本项目发现了汉黄芩苷调控血管正常化的作用和机制，为抗肿瘤血管治疗提供新的思路。