【摘要】 目的基于PI3K/AKT/mTOR信号通路,以人非小细胞肺癌耐顺铂细胞株A549/DDP为研究对象,探讨白藜芦醇联合顺铂对A549/DDP细胞增殖、凋亡、自噬的影响。方法以CCK-8法检测白藜芦醇、顺铂以及两药联用对A549/DDP细胞增殖的影响,并通过Isobologram分析两药联用的协同作用;将A549/DDP细胞分为空白对照组、白藜芦醇组、顺铂组、白藜芦醇+顺铂组;以Hoechst 33342染色和Annexin V/PI双染实验检测细胞凋亡率;免疫荧光实验检测自噬标志物LC3的表达;Western Blot实验检测各组细胞中凋亡相关蛋白、自噬标志物蛋白、PI3K/AKT/mTOR通路蛋白表达。结果与顺铂组比较,白藜芦醇与顺铂两药联用可以明显降低顺铂对A549/DDP细胞的IC50,Isobologram分析结果也表明二者联用对A549/DDP细胞具有协同抑制作用;与空白对照组、白藜芦醇组、顺铂组3组比较,白藜芦醇和顺铂联用可以明显增加A549/DDP细胞凋亡率(P<0.01),降低抗凋亡蛋白Bcl-2表达水平(P<0.01),上调凋亡蛋白Bax、Caspase 3表达水平(P<0.01),及增加自噬标志物蛋白LC3Ⅱ/Ⅰ表达水平(P<0.01),降低p62表达水平(P<0.01);此外Western Bolt实验结果表明两药联用可以明显下调p-PI3K、p-AKT,p-mTOR蛋白表达水平(P<0.01)。结论白藜芦醇与顺铂联用可通过PI3K/AKT/mTOR通路,促进A549/DDP细胞自噬诱导细胞凋亡。更多还原

【Abstract】 Objective From perspective of PI3 K/AKT/mTOR signaling pathway,the effects of resveratrol combined with cisplatin on proliferation, apoptosis and autophagy of A549/DDP cells were studied using cisplatin-resistant human non-small cell lung cancer cell line A549/DDP as a cell model. Methods CCK-8 assay was used to detect the effects of resveratrol,cisplatin and combination of the two drugs on the proliferation of A549/DDP cells,and the synergistic effect of the two drugs was analyzed by Isobologram. The A549/DDP cells were divided into blank control group,resveratrol group,cisplatin group and resveratrol + cisplatin group. The apoptosis rate of each group was detected by Hoechst 33342 staining and Annexin V/PI double staining. The expression of autophagy marker LC3 was detected by fluorescence assay. The expression of proteins related to apoptosis,autophagy marker and PI3 K/AKT/mTOR pathway of each group was detected by Western Blot assay. Results Compared with the cisplatin group,the combination of resveratrol and cisplatin could significantly reduce the IC50 of cisplatin on A549/DDP cells.Isobologram analysis also showed that the combination of the two drugs had synergistic inhibitory effect on A549/DDP cells. Compared with the blank control group, the resveratrol group and the cisplatin group, combination of resveratrol and cisplatin could significantly increase the apoptosis rate of A549/DDP cells(P<0.01),decrease the expression level of anti-apoptotic protein Bcl-2(P<0.01),up-regulate the expression levels of apoptotic protein Bax and Caspase 3(P<0.01),and increase the expression level of autophagy marker protein LC3Ⅱ/Ⅰ(P<0.01),decreased the expression level of p62(P<0.01). The results of Western Bolt assay showed that the combination of the two drugs could significantly down-regulate the protein expression levels of p-PI3 K,p-AKT and p-mTOR(P<0.01). Conclusion The combination of resveratrol and cisplatin can promote autophagy and induce apoptosis in A549/DDP cells through PI3 K/AKT/mTOR pathway.更多还原