TGF-β与内质网应激cross-talk促肺癌细胞上皮间质转化机制及益气除痰方干预作用研究

Metastasis is the main factor affecting the survival of lung cancer, and epithelial mesenchymal transition (EMT) plays an important role in invasion and metastasis. "Phlegm, deficiency" is the pathogenesis of lung cancer metastasis in traditional Chinese Medicine theory . Under the theory of"overall concept" and "preventive treatment of disease", prevention of metastasis should invigorate Qi and eliminate phlegm. The long-term clinical studies have also demonstrated that "Yiqi Chutan Formula" based on the principal have exact curative effect in the treatment of advanced lung cancer in elderly. Previous studies have shown that the formula can inhibit the metastasis of lung cancer. The previous proteomics research also found it can regulate the endoplasmic reticulum stress(ER stress) response and inhibit EMT. TGF-β is an important inducer of EMT. Literature studies suggest that ER stress is also an important inducer of EMT ，and it probably coordinate with TGF- β pathway to promote EMT. It put forward the hypothesis: Yiqi Chutan formula can regulate the interaction signal network between ER stress and TGF- β of lung cancer cell and inhibit EMT to futher hamper the metastasis of lung cancer.Study on the forward analysis and backward verification combination, will analyze the interaction between ER stress and TGF- β pathway in promoting the process of epithelial mesenchymal transition in lung cancer cells and the intervention of Yiqi Chutan Formula on this signal network of EMT. It can not only further clarify the mechanism of EMT, but also enrich the anticancer mechanism of traditional Chinese Medicine, and provide theoretical basis for the traditional Chinese Medicine " overall regulating" advantage.

肺癌远处转移是影响生存的主要因素,上皮间质转化（EMT）在肺癌侵袭转移中发挥重要作用。“痰、虚”是肺癌远处转移的病机，基于中医“整体观念”,抑制肺癌转移当益气除痰。“益气除痰方”经长期临床研究证实治疗晚期肺癌疗效确切。前期研究表明该方可抑制肺癌转移，蛋白质组学研究发现其可调控内质网应激反应及抑制EMT。TGF-β是EMT发生的“总开关”。文献研究提示内质网应激亦是诱导EMT的重要因素，其可能与TGF-β相互协同促进肿瘤细胞EMT。故提出假说：益气除痰方通过调控内质网应激与TGF-β相互作用的信号网络抑制肺癌细胞EMT抑制肺癌转移。研究将正向分析和反向验证相结合，探讨内质网应激与TGF-β通路相互作用促进肺癌细胞EMT的机制并在此基础上整体解析益气除痰方调控EMT的分子机制。目前中药复方调控肿瘤细胞EMT的机制研究相对匮乏，本项目可丰富中药抗癌机理，为中药复方“整体调节”优势提供新的依据。

肺癌的转移是影响临床疗效的重要因素，也是治疗失败和患者死亡的主要原因。上皮间质转化(Epithelial mesenchymal transition, EMT)是肿瘤细胞发生转移的关键机制之一。因此，通过抑制EMT从而达到抑制肿瘤细胞转移的目的是目前肿瘤研究的热点与难点之一。益气除痰方是经过国家科技部“十五”“十一五”项目验证的治疗肺癌有效方剂。. 本研究主要从探讨TGF-β与内质网应激在促进EMT进程中的相互作用机制，益气除痰方基于调控TGF-β与内质网应激互作机制抑制EMT的调控的分子网络两个方面进行研究。采用A549细胞进行相互作用机制研究及益气除痰方的分子机制的研究，采用裸鼠A549种植瘤模型进行益气除痰方的抗肺癌转移疗效验证及益气除痰方的分子机制验证，及采用了Batman系统进行了机制的验证。并采用超高效液相色谱技术分析出益气除痰方的有效成分。. 通过研究发现益气除痰方可以抑制A549裸鼠种植瘤的肺内转移；益气除痰方可以抑制A549细胞的迁移侵袭能力及EMT；并且益气除痰方可以通过抑制TGF-β下游的smad2/3信号通路、抑制内质网应激未折叠蛋白反应相关标志物如grp78、GRP94、CRT、ATF6a、ATF4来抑制EMT的发生；而益气除痰方的主要成分之一——贝母乙素可以抑制A549肺癌细胞的EMT，但与调控内质网应激反应可能无关。另外，TGF-β诱导EMT过程中激活了内质网应激，应用内质网应激抑制剂可以抑制TGF-β诱导的EMT；内质网应激的主要分子GPR78在EMT过程中发挥关键作用。益气除痰方有效成分包括迷迭香酸、贝母甲素、贝母乙素、β-谷甾醇等。. 本项目研究结果为揭示肿瘤细胞发生EMT的分子机制及补充新的理论及实验依据，为肺癌转移的有效防治提供新的治疗靶点。同时，本研究在中药复方研究领域进行了抑制EMT抗肺癌转移的研究，对中药抗癌机理理论的创新具有一定的补充及推动作用，为临床应用中医药防治肿瘤转移提供了有益的实验参考依据。通过中药单体与中药复方的对比研究，进一步揭示了中药复方的整体调节性的客观依据。