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线粒体DNA缺失细胞(ρ~0细胞)拮抗化疗药物诱导的凋亡,但其确切机制尚不明确。本研究探讨P-gp线粒体转位与人肝癌细胞(SK-Hepl)mtDNA缺失细胞(ρ~0SK-Hep1)多药耐药产生的关系。以SK-Hep1、ρ~0SK-Hep1和转线粒体细胞SK-Hep1Cyb为研究对象,CCK-8方法检测细胞对药物敏感性;AnnexinV/PI双染法及DAPI染色法检测细胞凋亡;Westernblot检测P-gp表达;激光共聚焦显微镜结合免疫荧光检测P-gP细胞内分布。结果显示,SK-Hep1、ρ~0SK-Hep1和SK-Hep1Cyb细胞对多柔比星(DOX)的IC_(50)分别为0.62±0.02μg/ml、4.93±0.17μg/ml和0.57±0.02μg/ml。SK-Hep1、ρ~0SK-Hep1和SK-Hep1Cyb细胞凋亡率分别为1 1.25%±1.36%、4.75%±0.98%和14.50%±1.57%,ρ~0SK-Hep1对细胞凋亡有明显抗性。Western blot检测发现ρ~0细胞内P-gP、Bax、Bcl-2表达增加,Bcl-2/Bax比值增加。免疫荧光共定位显示,ρ~0细胞线粒体内P-gP表达增加。结果表明,ρ~0细胞对化疗药物诱导的凋亡有明显抵抗,这种现象可能与ρ~0细胞P-gP、Bax、Bcl-2表达增加有关。ρ~0细胞P-gP线粒体转位可发挥外排泵作用将药物排出线粒体,改变化疗药物的亚细胞分布,减少细胞内药物浓度,使细胞产生多药耐药。
Mitochondrial DNA-deficient cells (ρ ~ 0 cells) antagonize chemotherapeutic drug-induced apoptosis, but its exact mechanism is not clear. This study was designed to investigate the relationship between P-gp mitochondrial translocation and multidrug resistance in mtDNA-deficient cells (ρ ~ 0SK-Hep1) in human hepatocellular carcinoma (SK-Hepl) cells. The cell chemosensitivity was detected by CCK-8 assay using SK-Hep1, ρ ~ 0SK-Hep1 and SK-Hep1Cyb cells. Apoptosis was detected by Annexin V / PI double staining and DAPI staining; -gp expression; laser scanning confocal microscopy combined with immunofluorescence detection of P-gP intracellular distribution. The results showed that IC 50 of doxorubicin (DOX) in SK-Hep1, ρ ~ 0SK-Hep1 and SK-Hep1Cyb cells were 0.62 ± 0.02μg / ml, 4.93 ± 0.17μg / ml and 0.57 ± 0.02μg / ml. The apoptosis rates of SK-Hep1, ρ ~ 0SK-Hep1 and SK-Hep1Cyb cells were 1 1.25% ± 1.36%, 4.75% ± 0.98% and 14.50% ± 1.57%, respectively. Sex. The results of Western blot showed that the expression of P-gp, Bax and Bcl-2 increased and the ratio of Bcl-2 / Bax increased in ρ ~ 0 cells. Immunofluorescence co-localization showed that the expression of P-gp in mitochondria of ρ ~ 0 cells increased. The results showed that ρ ~ 0 cells showed obvious resistance to chemotherapy-induced apoptosis, which may be related to the increased expression of P-gp, Bax and Bcl-2 in ρ ~ 0 cells. ρ ~ 0 cells P-gP mitochondrial translocation can play the role of efflux pump drug excretion of mitochondria, change the subcellular distribution of chemotherapy drugs, reduce intracellular drug concentration, the cells produce multi-drug resistance.