自噬抑制剂在内质网应激状态下对肝癌HepG2细胞和正常肝细胞L-02作用的差异  

The different function of autophagy inhibitors between HepG2 liver cancer cells and normal liver cells L-02 in the endoplasmic reticulum stress state

作　　者：李文成[1] 刘加涛[1] 高爽[1] 于瀚卿 吴圣[1] 范璐璐[1] 孙国平[1] 

机构地区：[1]安徽医科大学第一附属医院肿瘤科,合肥230022

出　　处：《安徽医科大学学报》2015年第7期884-888,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81272739);安徽省科技攻关项目(编号:12010402122)

摘　　要：目的研究自噬在内质网应激(ERS)状态下对肝癌Hep G2细胞和正常肝细胞L-02作用的差异。方法体外常规培养的人肝癌Hep G2细胞和正常肝细胞L-02,分别给予衣霉素(TM)单药和TM联合自噬抑制剂3-甲基腺嘌呤(TM+3-MA)或氯喹(TM+CQ)作用12、24、48 h后,采用噻唑蓝(MTT)法检测细胞活力变化,流式细胞术检测细胞凋亡率,Western blot法检测自噬蛋白LC3的变化。结果 TM可引起Hep G2细胞和L-02细胞死亡并呈时间依赖关系,3-MA或CQ均可增加TM对Hep G2细胞的生长抑制作用,24 h细胞存活率分别为TM+3-MA组(60%)、TM+CQ组(72%)、TM组(86%),差异有统计学意义(P<0.01);但对于L-02细胞,其存活率分别为83%、84%、83%,活力没有明显差异;流式细胞术显示TM+3-MA、TM+CQ和TM组对Hep G2细胞的凋亡率分别为15%、11%、7%,差异有统计学意义(P<0.01),但对L-02细胞,凋亡率分别为16%、17%、16%,未见明显差异;Western blot法结果显示TM作用引起两种细胞自噬增加,自噬抑制剂3-MA与CQ可引起两种细胞自噬作用减弱。结论自噬抑制剂(3-MA或CQ)均可显著增加TM对肝癌Hep G2细胞的生长抑制作用,但对正常肝细胞L-02的生长抑制作用差异无统计学意义。自噬在ERS状态下可对肝癌细胞的生存提供保护,但对正常肝细胞无保护作用。Objective To investigate the difference in function of autophagy inhibitors between HepG2 and L-02 cells in the state of endoplasmic reticulum stress. Methods The HepG2 cells and L-02 cells were routinely cul-tured in vitro and treated either with tunicamycin （ TM） or combination with the autophagy inhibitors 3-methylade- 〈br〉 nine （3-MA） or chloroquine （ CQ） . The cell viability was detected by MTT assay. Cell apoptosis was detected by flow cytometry. The change of autophagy-related protein LC3 was analysed by Western blot assay. Results MTT assay demonstrated that TM could time-dependently induce the death of HepG2 cells and L-02 cells, and the cell viability of HepG2 cells was significantly restrained when it was administrated in combination with 3-MA or CQ, the cell survival rates of 24 h were：3-MA＋TM （60%） , CQ＋TM （72%） , TM （86%） respectively, and the differ-ence was statistically significant （ P〈0. 01 ） . However, it had no significant effect with L-02 cells because of its cell survival rates of 24 h were：83%, 84% and 83%. Flow cytometry apoptosis experiment found that： TM＋3-MA, CQ＋TM and TM group on HepG2 cell apoptosis rates were 15%, 11% and 7% respectively, and the differ-ence was statistically significant（P〈0. 01）. But for L-02 cell group, the apoptosis rates were 16%, 17%, 16%which had no obvious difference. According to the results of Western blot, TM could cause increased autophagy, and autophagy inhibitor CQ could lead to increased autophagy tide. Conclusion The cell viability of HepG2 cells, not the L-02 cells, can be significantly restrained by TM combined with different autolysosome inhibitor 3-MA or CQ. So the autophagy can protect the hepatocarcinoma cell line HepG2 under the state of endoplasmic reticulum stress, and it can’ t provide the same protection to L-02 cell line.

关 键 词：自噬抑制剂 内质网应激 肝癌 肝正常细胞 

分 类 号：R735.7[医药卫生—肿瘤]