基于MACC1介导的HGF/c-Met-TWIST1/2通路探讨活血药干预肺癌干细胞血管生成拟态研究

The formation of angiogenesis mimicry (VM) has a key link with the process of tumor invasion and metastasis. Tumor stem cells may form the VM through the secretion of related factors and transformation of vascular endothelial cells. MACC1 mediated HGF/c- Met-TWIST1/2 pathway is an important signal when VM occurred. Early results showed blood activating herb can effectively suppress tumor angiogenesis by up-regulation of HIF-1 alpha and VEGF，down-regulation of E-cadherin. Whereas, VEGF and HIF-1, E-cadherin is the upstream and downstream genes of TWIST. Therefore, we can infer that the mechanism of blood activating herb anti lung cancer is through the regulation of TWIST related signaling pathways. The project will firstly investigate the effects of different types of blood drug on lung cancer vasculogenic mimicry, then sort CD133 + A549 cells with stem cell characteristics and observe their effects on lung cancer VM and its mechanism, then use Blood-activating drugs to act on the CD133+ A549 cells under different oxygen environment, and observe the effect of Blood-activating drugs on CD133+ A549 lung cancer cells and the expression of VM-related factors, to investigate the effect of Blood-activating drugs on VM in lung cancer whether related to the intervention of stem cells promoting secretion of VM related factors, in order to explore the mechanisms of Blood-activating drugs against lung cancer VM. Provide scientific basis for clinical reasonable application of Blood-activating drugs.

血管生成拟态（VM）是引起肿瘤侵袭和转移的关键环节，肿瘤干细胞可能通过分泌相关因子或直接转化为血管内皮细胞等方式形成VM。MACC1介导的HGF/c-Met-TWIST1/2通路是VM的重要信号通路。前期研究显示活血药可下调HIF-1α、VEGF的表达，上调E-cadherin的表达，有效抑制肿瘤血管生成。而HIF-1α、VEGF、E-cadherin为TWIST的上下游基因。因此，推断活血药抗肺癌机制可能通过对TWIST相关信号通路的调节而起效。本项目将分选具有干细胞特性的CD133+A549细胞并观察其对肺癌血管生成拟态的影响及机制，继而在不同氧环境下用活血药干预CD133+A549细胞，观察活血药对CD133+A549细胞与肺癌VM相关因子表达的影响，探讨活血药对肺癌VM的影响是否与对肺癌干细胞促VM相关因素的干预有关，从而探索活血药抗肺癌VM机制。为临床应用活血药提供科学依据。

本课题以活血药抑制肿瘤干细胞样细胞（CSLCs）血管生成拟态（VM）为方向，从体内、体外两方面探讨活血药在不同氧环境抑制CSLCs VM的作用及机制。研究通过无血清成球法富集A549 CSLCs，证实A549 CSLCs具有更强的迁移、克隆、小鼠移植瘤形成能力，且干细胞标志CD44+/CD24-/low的表达量高达80.4%；另通过体外小管形成实验证实A549 CSLCs较A549细胞有更强的促VM能力、VM相关分子HIF1α、VEGFA表达上调，并且经内皮细胞培养基诱导培养后能够向内皮细胞表型转化。通过体内模型验证川芎丹参对A549 CSLCs VM的影响，CD31/PAS双染法显示，无论常氧、乏氧环境，川芎、丹参、川丹联合均可抑制VM的形成；常氧环境下，丹参抑制VM相关因子VEGF、MMP-9、VE-Cadherin、FAK、EphA2、Integrinβ1蛋白的表达；川芎抑制促VM因子VEGF、MMP-9、EphA2的表达；川芎和丹参配伍可抑制促VM因子VEGF、MMP-9、VE-Cadherin的表达；乏氧环境下，川芎抑制促VM相关因子FAK、EphA2、Integrinβ1表达；丹参抑制VE-Cadherin、FAK表达；川芎和丹参则下调Integrinβ1蛋白。体外实验提示川芎嗪与丹参酮IIA均能抑制A549 CSLCs增殖与体外血管生成，且作用与药物浓度具有一定量效关系；常氧环境丹参酮IIA抑制A549 CSLCs FAK、cMET、Integrinβ、MMP-9、VEcad、TWIST1、TWIST2等VM相关分子表达，乏氧环境下丹参酮IIA抑制EphA2、HGF、HIF-1α、MMP-9、Integrinβ、TWIST2表达；常氧环境下，川芎嗪抑制cMET、MMP-9表达，乏氧环境川芎嗪抑制TWIST1/2，VE-Cadherin、MMP9、Integrinβ的表达。