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目的探讨人端粒酶逆转录酶(human telomerase reverse enzyme,hTERT)线粒体转位对肝癌细胞干性及耐药的影响。方法采用大剂量顺铂(CDDP)冲击、间歇诱导的方法诱导肝癌细胞HepG2、Huh7耐药,构建耐药细胞株HepG2/CDDP、Huh7/CDDP,CCK-8检测细胞耐药能力;Western blot检测细胞线粒体hTERT表达情况;激光共聚焦显微镜观察细胞线粒体膜电位;流式细胞术检测耐药细胞干性相关蛋白CD133、EpCAM表达情况;克隆形成实验检测细胞克隆形成能力;Western blot检测耐药细胞干性相关因子OCT-4蛋白表达水平变化。结果与亲本细胞HepG2[耐药指数(7.69±0.86)μg/m L及Huh7(7.18±0.35)μg/m L]相比,耐药细胞株对CDDP耐药指数明显增加[HepG2(41.16±0.42)μg/m L,Huh7(33.48±0.33)μg/m L,P<0.01],经顺铂(CDDP)诱导耐药细胞线粒体hTERT表达显著升高,线粒体膜电位增强,CD133~+细胞比例[HepG2(1.44±0.41),HepG2/CDDP(23.15±1.55),P<0.01]及EpCAM~+细胞比例[HepG2(0.85±0.10),HepG2/CDDP(3.96±0.10),P<0.01]增加,克隆形成能力增强,干性相关因子OCT-4蛋白表达水平增加。结论经顺铂诱导的肝癌耐药细胞线粒体hTERT转位显著增加,且具有明显的肿瘤干细胞特征。
Objective To investigate the effect of human telomerase reverse transcriptase (hTERT) mitochondrial translocation on the stem cell and drug resistance of hepatocellular carcinoma cells. Methods High-dose cisplatin (CDDP) shock and intermittent induction methods were used to induce HepG2 and Huh7 resistance in HepG2 cells. Drug-resistant cell lines HepG2 / CDDP, Huh7 / CDDP and CCK- Mitochondrial hTERT expression; laser scanning confocal microscopy of mitochondrial membrane potential; flow cytometry detection of drug-resistant cell-related protein CD133, EpCAM expression; clone formation assay to detect cell clonality; Western blot detection of drug-resistant cell dry Correlation factor OCT-4 protein expression level changes. Results Compared with HepG2 [(7.69 ± 0.86) μg / m L and Huh7 (7.18 ± 0.35) μg / m L], the resistance index of drug-resistant cell lines to CDDP was significantly increased [HepG2 (41.16 ± 0.42 (33.48 ± 0.33) μg / m L, P <0.01]. The expression of hTERT in mitochondria induced by cisplatin (CDDP) was significantly increased, the mitochondrial membrane potential was increased, and the proportion of CD133 ~ + cells [ HepG2 / CDDP (23.15 ± 1.55), P <0.01] and EpCAM ~ + cell ratio [HepG2 (0.85 ± 0.10) and HepG2 / CDDP (3.96 ± 0.10), P <0.01] The ability of formation increased, and the expression of OCT-4 protein increased. Conclusion The translocation of mitochondrial hTERT induced by cisplatin is significantly increased, and has obvious characteristics of tumor stem cells.