从Wnt信号途径介导的肿瘤相关巨噬细胞促血管新生探讨化痰祛瘀法干预肺癌的分子机制

Macrophages in tumor microenvironment play a key role in the growth and metastasis of tumor through angiogenesis mediated by the Wnt signal pathway."Reducing phlegm and dispelling stasis method " (RPDS) is the common and effective method for the main pathological factor "phlegm and static blood combined together" in the incident of lung cancer, and it can stabilize tumor, prolong survival and improve the patient's quality of life. Our previous study confirmed that "Gualou-Chishao"(GC) can inhibit angiogenesis and tumor growth,extend the survival period of the tumor-burdened rats,and regulate the hallmark cytokine expression of macrophages in the Wnt pathway. Thus, it is hypothesized that RPDS can regulate tumor-associated macrophages to inhibit angiogenesis and intervene growth and metastasis of tumor through the Wnt pathway. We take the angiogenesis of lung cancer as a starting point, the relationship between tumor-associated macrophages and Wnt pathway as target point. By using the way of 3-D angiogenesis model in vitro and lung cancer nude mouse model in vivo and a variety of molecular biology techniques to reveal the molecular mechanism of tumor-associated macrophages promoting angiogenesis mediated by the Wnt pathway and the target points of GC from multiple levels, such as overall level, cellular level, and molecular level. This project will reveal the mechanism of angiogenesis intervened by GC from the perspective of immune cell regulation in tumor microenvironment,providing the theoretical basis and experimental evidences for the treatment of lung cancer by RPDS.

肿瘤微环境中浸润的巨噬细胞通过Wnt信号通路介导的血管新生在肿瘤生长转移中起着关键作用。化痰祛瘀法是针对肺癌发病中主要病理因素“痰瘀互结”的常用和有效的治法，在稳定瘤体、延长生存期、改善生活质量等方面作用显著。前期研究显示“瓜蒌-赤芍”可阻抑血管生成，抑制肿瘤生长，延长荷瘤鼠生存期，并能调节Wnt通路相关的巨噬细胞标志性因子的表达。据此提出“化痰祛瘀法通过Wnt通路调控肿瘤相关巨噬细胞，阻抑血管新生，进而干预肺癌生长转移”的假说。课题以肺癌新生血管形成为切入点，以肿瘤相关巨噬细胞和Wnt通路之间的关系为靶点，采用体外三维新生血管形成和体内肺癌裸鼠模型，综合运用多种分子生物学技术，从整体、细胞、分子等多个层面揭示Wnt通路介导的肿瘤相关巨噬细胞促血管新生的分子机制及“瓜蒌-赤芍”的作用靶点。课题将从微环境免疫细胞调控角度揭示瓜蒌-赤芍干预血管新生的机理，为化痰祛瘀法治疗肺癌提供理论。

原发性支气管肺癌是目前最常见的恶性肿瘤之一，死亡率居首。新生血管的形成是肿瘤生长、侵袭转移的必要条件。肿瘤微环境中浸润的巨噬细胞，即肿瘤相关巨噬细胞 （tumor-associated macrophages，TAM）介导了新生血管形成，且与肺癌的治疗效果欠佳、预后不良密切相关。课题在中医整体观指导下，从微环境中肿瘤相关巨噬细胞与血管新生相互作用的角度出发，对“瓜蒌散如何通过调控微环境中M2巨噬细胞表型及功能阻抑血管新生”进行了研究。通过采用小鼠荷瘤模型以及体外巨噬细胞和内皮细胞共培养体系，综合运用多种分子生物学技术，我们的研究最终发现，Wnt/β-catenin信号通路在肿瘤相关巨噬细胞M2型极化过程中发挥重要作用，并能通过分泌IL-8、VEGF等细胞因子从而促进肿瘤的血管新生；瓜蒌散可以显著抑制Lewis肺癌小鼠皮下移植瘤的生长，显著降低原发灶肿瘤微环境中M2型巨噬细胞的表达，对M1型巨噬细胞无明显影响，并调控血管生成，降低相关标志物CD31的表达。进一步筛选并阐明了瓜蒌散有效单体血根碱阻抑M2型巨噬细胞介导血管新生的分子机制，发现血根碱可以通过抑制Wnt/β-catenin信号途径，阻抑M2型巨噬细胞极化，并抑制IL-8、VEGF的分泌最终阻抑肿瘤血管新生。从调控机体肿瘤相关免疫细胞功能、抗血管生成的新角度，阐释了瓜蒌散在防治肺癌中的分子机理，丰富化痰祛瘀法的现代科学内涵，为其临床应用提供理论依据，同时创新肺癌中医治法研究的思维与方法。