益气养肺法拮抗bFGF对肺癌微环境介导化疗耐药机制研究

Multidrug resistance（MDR） is the major cause of chemotherapy failure in lung cancer. MDR is closely related to cancer microenvironment. The major survival signal pathway of lung cancer is PI3k/Akt/mTOR. According to our prevous research, Supplement Energy and Nourish Lung Herbs (SENL) can inhibit chemotherapy drug resistance by down-regulate mitochondria membrane potential level and up-regulate Caspase activity. Recent research indicates that over-expression of basic fibroblast growth factor (bFGF) and multidrug resistance-associated protein (MRP) are important markers of primary chemotherapy drug resistance. SENL can significantly inhibit bFGF expression in lung cancer. The combination of SENL and chemotherapy, can enhance efficacy, reduce toxicity of chemotherapy and interfere with cancer microenvironment, which may play an important role in reversing MDR in lung cancr. In this research, determinated treatment based on the differentiation of symptoms and signs in lung cancer, clinical experience in Traditional Chinese Medicine (TCM), we select the drug resistance lung cancer cell line as research object, establish three dimensional cell culture, transplanted drug-resisance lung cancer cells in nude mice model, use molecular biology techniques, such as Real-Time PCR and Western blot. We investigate the influence of bFGF induced cancer microenvironment activation on lung cancer drug resistance both in vitro and in vivo, observe effect of SENL on bFGF associated cancer microenvironment, inhibition of lung cancer drug resistance and the effect on PI3k/Akt/mTOR signal transduction. Focused on improving chemotherapy efficacy in lung cancer, we will show and screen for effective TCM herbs for lung cancer drug resistance, search for mechanisms on lung cancer drug resistance inhibition by modern TCM. Therefore, further systemetical investigation on SNL herbs may lead to a considerable strategy for more successfl treatment both on TCM theory and experimental support.

多药耐药是老年肺癌化疗乏效主要原因，与肿瘤微环境密切相关，PI3k/Akt/mTOR是肺癌生存主要通路。我们前期研究发现益气养肺法拮抗肺癌化疗耐药，下调线粒体膜电位水平，上调半胱天冬酶活性。近期实验显示碱性成纤维生长因子(bFGF)和多药耐药相关蛋白(MRP)表达增强, 作为肺癌原发性耐药重要标志物，益气养肺方药明显抑制肺癌bFGF表达。益气养肺法联合化疗增效减毒，干预肿瘤微环境可能在拮抗肺癌耐药发挥重要作用。本研究遵循中医辨证施治及临证经验，以肺癌耐药为研究对象，构建荷瘤裸鼠模型，运用三维细胞培养、定量PCR和Western blot等手段，在细胞和动物水平研究bFGF对肿瘤微环境活化介导肺癌耐药作用, 益气养肺法及方药对肺癌bFGF和肿瘤微环境影响,并拮抗肺癌耐药PI3k/Akt/mTOR信号机制，为阐明益气养肺法具有时代中医药内涵抗肺癌耐药机理，做出有效探索和理论及实验支撑。

本项目根据任务书计划，顺利完成益气养肺法拮抗bFGF对肺癌微环境介导化疗耐药机制研究，主要研究工作成果：.（1）研究显示bFGF表达与非小细胞肺癌（NSCLC）临床分期、分化程度、淋巴结转移相关，同时发现bFGF和CCL18共同促进肺癌浸润、侵袭及转移；bFGF诱导肺癌发生上皮间质转化（EMT），增强侵袭迁移和对顺铂耐药，bFGF单抗通过逆转EMT，抑制肺癌侵袭能力，提高对顺铂的敏感性。.（2）荟萃分析表明益气养肺法沙参麦冬汤联合对恶性肿瘤的增效减毒作用优于单纯化疗。益气养肺法复方联合化疗治疗非小细胞肺癌，提高观缓解率、1年生存率、生存质量，改善中医证候，降低骨髓抑制。.（3）采用高效液相色谱法同时测定益气养肺方主要成分Rg1、Rb1、Rg3、汉防己甲素、黄芪甲苷、麦冬皂苷D的含量，均达到药典标准，为益气养肺方的质量控制提供实验依据。.（4）益气养肺方NSENL作用肺癌耐药细胞后， P-糖蛋白表达率下调，MDR1、bFGF、FGFR mRNA表达明显下调，研究NSENL对耐顺铂人肺腺癌A549/DDP裸鼠体内移植瘤生长的影响，通过NSENL对A549/DDP裸鼠体内移植瘤生长的抑制实验显示， NSENL联合化疗对A549/DDP裸鼠体内移植瘤的生长具有显著地抑制作用；进一步实验表明SENL具有降低MRP1蛋白及mRNA表达，下调GST表达，并诱导肺癌凋亡和抑制肿瘤新生血管的作用；NSENL可降低bFGF、FGFR表达, qPCR结果表明SENL降低bFGF、FGFR mRNA水平，抑制Akt、mTOR磷酸化水平，达到逆转肺癌化疗耐药作用。构建稳定高表达FGFR1基因 A549/FGFR1细胞株，深入研究NSENL联合bFGF单抗逆转肺癌耐药。.（5）荟萃分析EML4-ALK融合基因在肺癌患者表达的分子特点，及miR133a靶向肺癌微环境MMP-14抑制肺癌增殖，迁移与侵袭； 研究bFGF单抗协同洛铂降低肺腺癌细胞侵袭和转移，与抑制MMP-2和MMP-9表达有关；bFGF单抗逆转MCF-7/ADM多药耐药性，下调MDR1/P-gp表达。.（6）本项目形成的主要成果有①发表学术论文12篇，其中SCI收录6篇，并在世界抗癌大会和世界肺癌大会作口头报告，②培养博士研究生2名，硕士研究生5名。.（7）为针对性筛选逆转肺癌耐药的有效中药复方提供研究依据和有效探索。