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目的 :探讨榄香烯乳(elemene)逆转人肺腺癌耐顺铂(cisplatin,DDP)细胞A549/DDP的耐药性及可能的作用机制。方法 :采用MTT法检测榄香烯乳单药作用时的细胞毒作用及与DDP联合作用对A549/DDP细胞耐药的逆转作用;Hoechst 33342法进行细胞核染色,荧光显微镜下观察细胞形态的改变;FCM检测细胞凋亡率;蛋白质印迹法检测细胞质中细胞色素C(cytochrome C,cyt C)、pro-caspase-3、caspase-3和抗凋亡因子B细胞淋巴瘤-2(B cell lymphoma-2,Bcl-2)家族蛋白表达的情况。结果 :不同浓度榄香烯乳对A549/DDP细胞均有一定的抑制作用,呈时间-剂量依赖性效应,联合DDP能提高耐药细胞株A549/DDP对DDP的敏感性而逆转其耐药性。Hoechst 33342法和FCM法检测结果显示,榄香烯乳联合DDP能更大程度上诱导A549/DDP细胞的凋亡,提高耐药细胞的凋亡率,同时还上调A549/DDP细胞质中cyt C、caspase-3和Bad蛋白的表达,而下调pro-caspase-3和Bcl-2蛋白的表达。结论 :榄香烯乳能逆转A549/DDP细胞耐药性可能与其损伤线粒体膜,使线粒体释放cyt C到细胞质、同时活化caspase-3、上调促凋亡蛋白Bad和下调抗凋亡蛋白Bcl-2表达以启动凋亡路径有关。
AIM: To investigate the drug resistance and possible mechanism of elemene in reversing human lung adenocarcinoma A549 / DDP cells. Methods: MTT assay was used to detect the cytotoxic effect of elemene and the reversal of multidrug resistance in A549 / DDP cells induced by elemene. The cell morphology was observed by Hoechst 33342 staining and the cell morphology was observed by fluorescence microscopy. FCM was used to detect the apoptosis rate. Western blotting was used to detect the expression of cytochrome C, pro-caspase-3, caspase-3 and B cell lymphoma-2 , Bcl-2) family protein expression. Results: Different concentrations of elemene inhibited A549 / DDP cells in a time-and dose-dependent manner. Combination of DDP and DDP could improve the sensitivity of drug-resistant cell line A549 / DDP to DDP and reverse its drug resistance . The results of Hoechst 33342 assay and FCM assay showed that elemene combined with DDP could induce apoptosis of A549 / DDP cells to a greater extent and increase the apoptosis rate of A549 / DDP cells. Meanwhile, it also upregulated cyt C, caspase-3 and Bad protein expression, and down-regulation of pro-caspase-3 and Bcl-2 protein expression. CONCLUSION: Elemene can reverse the drug resistance of A549 / DDP cells and impair the mitochondrial membrane, release mitochondria cyt C to the cytoplasm, activate caspase-3, up-regulate pro-apoptotic protein Bad and down-regulate anti-apoptotic protein Bcl-2 Expression is related to the initiation of apoptosis pathways.