培土生金法对肺癌顺铂耐药Wnt/β-catenin信号通路干预的分子机制研究

Traditional Chinese Medicine (TCM) holds that the occurrence of cisplatin resistance of lung cancer is mainly due to “insufficiency of healthy-Qi”, “deficiency of spleen-Qi ”. Early research had shown that the mechanism of cisplatin resistance of lung cancer was closely related to inhibiting apoptosis induced by excessive activation of PI3K/Akt signaling pathway. Method of strengthening the lung by way of reinforcing the spleen could intervene the process effectively. Wnt/β-catenin signaling pathway as the downstream pathway of PI3K/Akt signaling pathway is the key to cell apoptosis. So we speculate that Wnt/β-catenin signaling pathway involves in cisplatin resistance of lung cancer. From the perspective of the overall concept of TCM, with the Wnt/β-catenin signaling pathway as the entry point, with the model of cisplatin resistance of lung cancer and A549/DDP cells as the study object, by techniques of flow cytometry, immunofluorescence, real-time RT-PCR, report gene, Western blotting and RNA interference, etc, with intervention of Chinese medicine of strengthening the lung by way of reinforcing the spleen, the project studies that the method of strengthening the lung by way of reinforcing the spleen effects on expressions of related signaling proteins and target gene, and clarifies the nature of “strengthening the lung by way of reinforcing the spleen”. It provides the referable experimental basis on filtering Chinese medicine which improves cisplatin resistance of lung cancer through selectively intervening Wnt/β-catenin signaling, and a new idea or theory for the pathogenesis and therapy of lung cancer resistance.

中医学认为肺癌顺铂耐药的发生主要是正气虚损、脾气不足所致。前期研究表明肺癌顺铂耐药机制与PI3K/Akt信号通路过度活化所致细胞凋亡抑制密切相关，培土生金法可有效干预其发生。PI3K/Akt信号通路下游的Wnt/β-catenin信号通路是细胞凋亡过程的关键，推测Wnt/β-catenin信号通路也参与肺癌顺铂耐药。本项目从中医学整体观出发，以Wnt/β-catenin信号通路为切入点，以肺癌顺铂耐药动物模型和A549/DDP细胞为研究对象，运用流式细胞术、免疫荧光法、实时RT-PCR、报告基因法、蛋白质印迹法、RNA干扰等技术，以培土生金中药进行干预，探讨培土生金法对肺癌顺铂耐药的相关信号蛋白及其靶基因表达的影响，阐明“培土生金”的本质。为筛选通过选择性干预Wnt/β-catenin信号转导改善肺癌耐药的中医药提供可参考的实验依据，同时为拓展肺癌耐药的病机与治法理论提供新的思路与方法。

本项目提出“肺癌顺铂耐药机制与Wnt/β-catenin信号通路过度活化所致细胞凋亡抑制密切相关，培土生金法可有效干预其发生”的假设。它以Wnt/β-catenin信号通路为切入点，以肺癌顺铂耐药动物模型和A549/DDP细胞为研究对象，运用流式细胞术、免疫荧光法、实时RT-PCR、蛋白质印迹法等技术，以培土生金中药进行干预，探讨培土生金法对肺癌顺铂耐药的相关信号蛋白及其靶基因表达的影响，阐明“培土生金”的本质。实验结果显示在顺铂持续的作用下，培土生金中药可有效抑制移植瘤的质量、体积，提高抑瘤率；可有效抑制A549/DDP细胞的生长，促进细胞凋亡；可显著下调移植瘤和A549/DDP细胞LRP、MRP、P-gp的表达；可显著下调移植瘤和A549/DDP细胞p-GSK-3βser9、β-catenin及survivin的表达。上述结果证实了培土生金法可通过抑制Wnt/β-catenin信号转导通路而有效干预肺癌顺铂耐药的发生。这为筛选通过选择性干预Wnt/β-catenin信号转导改善肺癌耐药的中医药提供可参考的实验依据，同时为拓展肺癌耐药的病机与治法理论提供新的思路与方法。