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目的探索耐阿霉素的乳腺癌细胞株MCF-7/Adr凋亡的可能途径。方法采用耐药株MCF-7/Adr和野生株MCF-7/wt2种细胞株进行对比,以噻唑蓝分析(MTT)法探索H2O2导致其线粒体功能下降的浓度,观察细胞形态学和线粒体超微结构的改变。应用DNA凝胶电泳、AnnexinⅤ和PI双标记流式细胞术检测细胞凋亡发生,Rh123共聚焦显微镜检测线粒体膜电位和P-gp功能,Western blot检测细胞色素C(Cyto C)的释放状况。结果4mmol/L H2O2引起MCF-7/Adr线粒体整体功能不可逆的进行性下降,线粒体空化,嵴肿胀。MCF-7/Adr凋亡率(96.52%)显著高于MCF-7/wt(18.57%,P<0.01)。凋亡DNA电泳仅见800、1600bp2个分子片段。MCF-7/Adr线粒体数量较MCF-7/wt减少,MCF-7/Adr膜电位下降。4mmol/L H2O2处理使MCF-7/Adr线粒体Cyto C释放至细胞质,MCF-7/wt仅少量Cyto C释放。结论P-gp阳性细胞因其线粒体的结构和功能改变的特点,较其野生株细胞更易经线粒体途径凋亡。提示以线粒体为靶点的药物有助于克服与P-gp表达有关的多药耐药。
Objective To explore the possible pathways of adriamycin-resistant breast cancer cell line MCF-7 / Adr. Methods MTT assay was used to investigate the effect of H2O2 on mitochondrial function decline. The morphological and mitochondrial ultrastructure of MCF-7 / Adr cells were observed by MTT assay. Structural changes. Cell apoptosis was detected by DNA gel electrophoresis, Annexin Ⅴ and PI double labeling flow cytometry. Mitochondrial membrane potential and P-gp function were detected by Rh123 confocal microscope. The release of Cyto C was detected by Western blot. Results 4 mmol / L H 2 O 2 caused irreversible decline of mitochondrial MCF-7 / Adr mitochondrial function, cavitation of mitochondria and swelling of cristae. The apoptosis rate of MCF-7 / Adr (96.52%) was significantly higher than that of MCF-7 / wt (18.57%, P <0.01). Apoptotic DNA electrophoresis saw only 800, 1600bp2 molecular fragments. MCF-7 / Adr mitochondrial MCF-7 / wt MCF-7 / wt decreased, MCF-7 / Adr membrane potential decreased. Treatment with 4mmol / L H 2 O 2 resulted in the release of Cyto C to the cytoplasm of MCF-7 / Adr mitochondria and only a small amount of Cyto C release from MCF-7 / wt. Conclusion The P-gp positive cells are more vulnerable to mitochondrial apoptosis than wild-type cells because of their altered mitochondrial structure and function. Suggesting that mitochondria as a target drug can help overcome the multi-drug resistance associated with P-gp expression.