摘要目的：探讨蟾毒灵对肝细胞生长因子（ HGF）诱导的阿法替尼耐药肺癌细胞H1975生长的影响，并分析其可能的作用机制。方法采用外源性HGF和重组HGF腺病毒转染内源性HGF诱导的方法建立阿法替尼耐药的肺癌细胞H1975，应用四甲基偶氮唑蓝（ MTT）法检测蟾毒灵、阿法替尼及联合用药对阿法替尼耐药细胞H1975增殖的抑制作用，Transwell实验检测药物对阿法替尼耐药细胞H1975侵袭能力的影响，酶联免疫吸附试验（ ELISA）法检测转染后H1975细胞分泌的HGF水平以及药物对HGF分泌水平的影响，Western blot检测药物干预后EGFR、cMet信号通路及上皮细胞?间质转化（ EMT）相关蛋白的表达。结果 MTT结果显示，在外源性和内源性HGF刺激下，阿法替尼对H1975细胞生长无抑制作用，经蟾毒灵与阿法替尼联合处理后，可明显抑制肿瘤细胞的生长。Transwell实验结果显示，在外源性HGF作用下，阿法替尼组的穿膜细胞数为（118．92±37．29）个；蟾毒灵组的穿膜细胞数为（88．84±19．53）个；阿法替尼与蟾毒灵联合组的穿膜细胞数为（48．98±11．43）个，与阿法替尼组和蟾毒灵组比较，差异均有统计学意义（均P＜0．05）。 ELISA结果显示，H1975／HGF细胞（转染重组HGF腺病毒的H1975细胞）分泌高水平的HGF，蟾毒灵和阿法替尼干预后，H1975／HGF细胞分泌HGF水平差异无统计学意义（P＞0．05）。 Western blot结果显示，蟾毒灵联合阿法替尼能明显下调阿法替尼耐药细胞中 p?EGFR、p?cMet、p?AKT、p?ERK、vimentin 和 snail 蛋白的表达，上调E?cadherin蛋白的表达。结论蟾毒灵联合阿法替尼可显著抑制阿法替尼耐药细胞H1975的增殖，阻止H1975肺癌细胞侵袭；蟾毒灵逆转 HGF 诱导的 H1975肺癌细胞对阿法替尼耐药，可能与阻断cMet／PI3K／AKT、cMet／MAPK／ERK信号通路以及阻止EMT进程有关。

abstractsObjective To investigate the effects of bufalin in reversing hepatocyte growth factor ( HGF)?induced resistance to afatinib in H1975 lung cancer cells, and explore its possible mechanism. Methods The afatinib?resistant H1975 lung cancer cells ( H1975AR) were induced by exogenous HGF and transfected with recombinant adenoviral vector Ad?HGF?GFP. The cytostatic effects of bufalin, afatinib and bufalin plus afatinib on H1975AR cells were evaluated by MTT assay. The impact of combined therapy with bufalin and afatinib on invasion of H1975AR cells was determined by transwell migration assay. The concentrations of HGF in the culture supernatants of H1975/Vec and H1975/HGF cells were determined by ELISA. The expression of EGFR, cMET and EMT signal pathway?related proteins in H1975AR cells treated with bufalin, afatinib and bufalin plus afatinib were detected by Western blot. Results The results of MTT assay showed that afatinib did not inhibit the growth of H1975 cells, but after 72 h of the combined treatment with bufalin and afatinib and in the presence of HGF, the growth rate of H1975 cells was (38.67±8.76)%, significantly lower than the growth rate of (63.45±12.65)% in the H1975 cells treated with HGF alone (P<0.05) . The results of transwell migration assay showed that in the presence of HGF, afatinib plus bufalin combination therapy markedly decreased the number of invaded H1975 cells through the Matrigel chamber&nbsp;(48.98±11.43), significantly lower than the 118.92±37.29 of afatinib?treated or the 88.84±19.53 of bufalin?treated cells (P<0.05 for all). The result of ELISA showed that H1975/HGF cells secreted high levels of HGF, and afatinib and bufalin had no effect on the HGF secretion in H1975/HGF cells. The results of Western blot analysis showed that the expression of p?EGFR, p?cMet, p?AKT, p?ERK, vimentin and snail in H1975AR cells treated with bufalin puls afatinb was down?regulated markedly, and the expression of E?cadherin was up?regulated markedly. Conclusions Combination of bufalin and afatinib strongly inhibits the growth of H1975AR lung cancer cells and decreases their invasion ability. The possible mechanism of combined treatment with bufalin and afatinib may be related to the blocking of cMet/PI3K/AKT and cMet/MAPK/ERK pathways and inhibiting of epithelial?mesenchymal transition.