基于“TAMs极化重编程诱导血管正常化”探究软坚散结胶囊抗脑胶质瘤的作用与机制

Inducing the normalization of tumor vasculature to improve tumor microenvironment and increase drug delivery efficacy is the promising therapeutic strategy for glioma. Previously, we have found that Ruan-jian San-jie capsule can targeted vascular in glioma, however, the mechanism is urgently to be explored. In terms of vascular normalization mediated by tumor-associated macrophages polarization reprogramming (skewing tumor-associated macrophages polarization away from the M2- to a tumor-inhibiting M1-like phenotype), the purpose of this project is to further study the therapeutic mechanism of Ruan-jian San-jie capsule for glioma. Content: molecular biology, transmission electron microscope, perfusion of FITC-dextran combined with confocal laser three dimensional reconstruction technology, et al. would be applied to detect the state of macrophage polarization, the levels of HRG, as well as vascular structure and function in the experimental C6 gliomas rat brain. They would be observed to assess therapeutic effects of Ruan-jian San-jie capsule. In the in vitro experiment, the effects of conditioned medium of macrophages on the biological behavior of pericyte/endothelial cells would be detected to further evaluate the effects of macrophages polarization reprogramming on vascular normalization. Significance: by elucidating the mechanism, it would provide scientific basis for the clinical application of Ruan-jian San-jie capsule in glioma. This research would provide new idea and method for searching candidate drugs of glioma.

诱导高度无序的血管恢复正常化，以改善肿瘤微环境及为药物运输提供通道是抗胶质瘤的重要策略。前期研究证实软坚散结胶囊可干预脑胶质瘤的血管结构和功能，本项目拟从TAMs极化重编程（远离M2型，朝向M1型极化）诱导血管结构和功能正常化的角度，进一步探究软坚散结胶囊抗脑胶质瘤的具体作用形式及机制。内容：体内实验通过分子生物学、透射电镜、FITC-dextran灌注结合激光共聚焦三维血管重建等技术观察脑胶质瘤大鼠脑内TAMs极化情况、血管结构和功能状态，并检测与TAMs极化重编程密切相关的HRG蛋白的表达明确软坚散结胶囊的影响；细胞实验通过检测不同极化状态巨噬细胞的条件培养液对周细胞以及内皮细胞生物行为学影响，从体外水平进一步明确巨噬细胞极化状态对血管正常化的影响及软坚散结胶囊的干预作用。意义：通过揭示软坚散结胶囊抗脑胶质瘤的机理，为其临床应用提供科学依据，为脑胶质瘤寻找候选药物提供新的思路与方法。

肿瘤微环境下结构和功能高度异常的血管不仅可以导致肿瘤发生转移，同时也阻碍了化疗药物的分布和利用。诱导高度无序的血管恢复正常化，以改善肿瘤微环境及为药物运输提供通道是抗脑胶质瘤的重要策略。肿瘤相关巨噬细胞（TAMs）具有高度的异质性，肿瘤微环境中M2型TAMs，能够促进紊乱的血管网络形成，继而促进肿瘤生长。证据显示，促进TAMs由M2型向M1型极化转化可能是驱动脑胶质瘤血管正常化的关键因素。软坚散结胶囊功能化痰软坚散结、活血解毒。临床上，软坚散结胶囊治疗脑胶质瘤取得了良好的疗效。有必要在基础实验方面开展对其作用机制的进一步探索。本项目研究内容包括：1. 体内实验：软坚散结胶囊介导的TAMs极化状态改变对脑胶质瘤血管结构及功能正常化的影响、对脑胶质瘤组织乏氧状态及瘤体内化疗药物替莫唑胺的输送的影响及其作用机制。2. 体外研究：构建巨噬细胞M2型极化状态模型，利用软坚散结胶囊含药血清为干预工具，围绕HIF-1α/VEGF信号通路，从体外水平进一步明确巨噬细胞极化重编程对血管正常化的影响及软坚散结胶囊的干预作用。本研究获得了以下几个研究结果：1. 软坚散结胶囊能够促进脑胶质瘤血管结构及功能的正常化；2. 软坚散结胶囊诱导的血管正常化能够影响胶质瘤组织乏氧状态及瘤体内TMZ的输送；3.软坚散结胶囊通过影响TAMs由M2型向 M1型转化，而促进脑胶质瘤血管正常化；4. 软坚散结胶囊通过抑制HIF-1α/VEGF信号通路影响TAMs极化重编程诱导的血管正常化。本研究将进一步阐明软坚散结胶囊抗脑胶质瘤的现代生物学基础，为其临床应用提供新的科学依据，为中医药治疗脑胶质瘤的研究提供新的思路。