扶正祛邪方调控肿瘤相关巨噬细胞介导的上皮间质转化预防肺癌转移的分子机理研究

Metastasis is the major cause of death in patients with lung cancer.M2 macrophages showed dominant expression in lung cancer microenvironment, and is related to the key events in early metastasis of epithelial to mesenchymal transition (EMT) closely, and there was a negative correlation with the survival in patients with lung cancer. Early studies suggested that FuzhengQuxie prescription could prolong survival of patients with lung cancer, inhibit tumor growth and metastasis, block the occurrence of EMT process, and inhibit the ability of M2 macrophages induced factor TGF-β in the microenvironment. Here, we postulated that FuzhengQuxie Compound can stop EMT process to prevent and control the invasion and metastasis of lung cancer through regulating the phenotype and function of M2 macrophages in lung cancer microenvironment. The subject will observe the expression of M2 macrophages, specific markers of EMT process and the circulating tumor cells under the treatment of the Compound and reveal the molecular mechanism of mediating the occurrence of EMT process by M2 macrophages and the treatment target for the Compound from multiple layers, such as whole, cell and molecules levels by taking advantage of Lewis lung cancer model and co-culture system of macrophages with lung cancer cells in vitro, adopting microfluidic technology, imaging technology in vivo, flow cytometry, microarray analysis, RNA interference technology and so on. The subject will reveal the molecular mechanism of EMT occurrence and the prevention of metastasis from the perspective of immune cells regulation under intervention of this FuzhengQuxie Compound in lung cancer microenvironment, provide the basis for clinical application.

转移是肺癌患者死亡的首要原因。肺癌微环境中呈优势表达的M2巨噬细胞与转移发生的早期关键事件-上皮间质转化（EMT）密切相关，且与患者生存期成负相关。前期研究显示扶正祛邪方可延长肺癌患者生存期、抑制肿瘤生长和转移发生、阻断EMT过程，并对微环境中M2巨噬细胞诱导因子TGF-β等具有抑制作用。据此提出"扶正祛邪方是否通过调控微环境中M2巨噬细胞表型及功能阻抑EMT过程进而防治肺癌侵袭转移"的假说。课题采用Lewis肺癌模型及体外巨噬细胞和肺癌细胞共培养体系，综合运用微流控技术、活体成像技术、流式细胞术、基因芯片、RNA干扰等技术，观察扶正祛邪方对M2巨噬细胞、EMT及循环肿瘤细胞表达的影响，从整体、细胞、分子等多个层次揭示M2巨噬细胞介导EMT发生的分子机制以及"扶正祛邪方"的作用靶点。课题将从肿瘤微环境免疫细胞调控角度揭示该方干预EMT发生和预防转移的分子机理，为临床推广应用提供依据。

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，转移是肺癌患者死亡以及疗效能以提高的首要原因。肺癌微环境中呈优势表达的M2巨噬细胞与转移发生的早期关键事件—上皮间质转化（EMT）密切相关，且与患者生存期成负相关。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肿瘤细胞相互作用的角度出发，对“扶正祛邪方如何通过调控微环境中M2巨噬细胞表型及功能阻抑EMT过程防治肺癌”进行了研究。通过采用Lewis肺癌模型以及体外巨噬细胞和肺癌细胞共培养体系，综合运用多种分子生物学技术，我们的研究最终发现，肿瘤相关巨噬细胞可能通过分泌IL-10/pStat3途径促进肿瘤细胞上皮间质转化，从而促进肿瘤的侵袭转移；扶正祛邪方可以显著抑制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EMT相关标志物的表达，升高E-cadherin的表达，降低N-cadherin、Vimentin、snail的表达。进一步筛选并阐明了扶正祛邪方活性组分愈创醇抑制肿瘤相关巨噬细胞介导EMT的分子机制，发现愈创醇可以通过抑制肿瘤相关巨噬细胞分泌IL-10，抑制STAT3信号通路，从而阻抑肺癌细胞上皮间质转化的发生。从调控机体肿瘤相关免疫细胞功能、提高自身抗癌能力的角度，阐释了扶正祛邪方在肺癌防治中的分子机理，为其临床应用提供理论依据，创新中医药复方治疗肺癌的研究思维和方法，有助于进一步改善肺癌的总体预后。