体外肺癌上皮-间充质转化三维模型的建立及其对顺铂抵抗的机制研究

目的:探索转化生长因子β1(transforming growth factor-β1,TGF-β1)诱导三维培养肺癌细胞发生上皮-间充质转化(epithelial-mesenchymal transition,EMT)及对顺铂抵抗的相关机制。方法:用荧光倒置显微镜、扫描电镜及激光共聚焦观察人非小细胞肺癌细胞系95D在TGF-β1刺激前后的形态及蛋白表达变化,Western blot检测三维培养95D细胞在TGF-β1刺激前后相关蛋白表达变化,MTT法检测95D细胞发生EMT前后对顺铂的敏感性变化。结果:在三维培养条件下,95D细胞在TGF-β1刺激后,细胞球出现塌陷、细胞离散、迁移等现象,细胞球之间互相融合。激光共聚焦结果显示95D细胞在TGF-β1刺激后,E-cadherin蛋白表达无变化,N-cadherin和vimentin蛋白表达显著上调。Western blot结果显示,在TGF-β1刺激后,95D细胞的E-cadherin蛋白表达下调(P<0.05),N-cadherin和vimentin蛋白表达上调(P<0.05),p-AKT和p-mTOR蛋白水平升高(P<0.05)。而LY294002和rapamycin可逆转TGF-β1诱导的上述蛋白表达(P<0.05)。MTT结果显示,TGF-β1刺激组对顺铂的敏感性显著低于普通三维培养组(P<0.01),而LY294002和rapamycin增强TGF-β1刺激后95D细胞对顺铂的敏感性(P<0.01)。结论:TGF-β1诱导三维培养肺癌细胞发生EMT及对顺铂抵抗,可能是通过激活PI3K/AKT/mTOR信号通路实现的。

A 3D cell culture system to study epithelial-mesenchymal transition and drug resistance of lung cancer

AIM:To explore the molecular mechanism of transforming growth factor-β1(TGF-β1)-induced epithelial-mesenchymal transition(EMT)and cisplatin resistance in three-dimensionally cultured lung cancer cells.METHODS:Under three-dimensional culture condition,the morphological changes and protein expression changes of human non-small-cell lung cancer 95D cells were observed by inversed fluorescence microscopy,scanning electron microscopy,laser scanning confocal microscopy and Western blot before or after TGF-β1 stimulation.The cisplatin sensitivity was determined by MTT assay.RESULTS:Under the three-dimensional culture condition,the structure of 95D cell spheroids after TGF-β1 stimulation collapsed,the cells were dispersed and migrating,and the spheroids merged with each other.The results of laser confocal microscopy showed that E-cadherin protein expression in the 95D cells did not changed after TGF-β1 stimulation,and the protein expression of N-cadherin and vimentin was significantly up-regulated.The results of Western blot showed that the expression of E-cadherin was down-regulated after TGF-β1 stimulation,and the protein levels of N-cadherin,vimentin,phosphorylated AKT and phosphorylated mTOR were up-regulated.LY294002 and rapamycin reversed TGF-β1-induced expression of the above proteins.The results of MTT assay showed that TGF-β1 reduced the sensitivity of three-dimensionally cultured 95D cells to cisplatin,while LY294002 and rapamycin reversed the cisplatin resistance of the 95D cells stimulated by TGF-β1.CONCLUSION:TGF-β1 induces the EMT and cisplatin resistance of three-dimensionally cultured lung cancer cells through the activation of PI3K/AKT/mTOR signaling pathway.