摘要
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 determine the effect of mitochondrial translocation of human telomerase reverse enzyme （hTERT） on the stemness and drug-resistance of hepatocellular carcinoma （HCC） cells. Methods Human HCC cell lines HepG2 and Huh7 were exposed intermittently to cisplatin （CDDP） to obtain cisplatin-resistant cell lines HepG2/CDDP and Huh7/CDDP. CCK-8 assay was employed to detect cell sensitivity to CDDP. Western blotting was adopted to detect hTERT protein level in the mitochondria, and mitochondrial membrane potential was measured by confocal laser scanning microscopy. Flow cytometry was used to detect the expression of cell stemness-related proteins CD133 and EpCAM, and the colony formation ability of the cells was assessed with colony formation assay; Western blotting was used to detect the expression level of stemness-related factor OCT-4. Results The resistance indexes of CDDP-resistant HepG2 and Huh7 cells were significantly increased compared with their parental cells （HepG2： 41.16±0.42 vs 7.69±0.86 μg/mL; Huh7： 33.48±0.33 vs 7.18±0.35 μg/mL, P〈0.01）. The CDDP=resistant cells exhibited significantly increased mitochondrial hTERT expression and mitochondrial membrane potential with increased proportions of CD133＋ cells （HepG2： 1.44±0.41, HepG2/CDDP： 23.15±1.55, P〈0.01） and EpCAM＋cells （HepG2： 0.85±0.10, HepG2/CDDP： 3.96±0.10, P〈0.01）. The resistant cells also showed significantly enhanced colony formation ability and increased expression of OCT-4 protein. Conclusion The translocation of mitochondrial hTERT is significantly increased in CDDP-resistant cells, which possess obvious phenotypes of tumor stem cells.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2017年第8期781-786,共6页
Journal of Third Military Medical University
关键词
端粒酶逆转录酶
线粒体
肿瘤干细胞
干性
耐药
human telomerase reverse enzyme
mitochondria
cancer stem cells
stemness
drug-resistance
作者简介
[通信作者]凌贤龙，电话：（023）68774204，E-mail：lingxianlong@yeah．net
