自噬机制在雷帕霉素对肝癌细胞化疗增敏中的研究

Mechanism of autophagy in enhanced effects on epirubicin-based chemotherapy by rapamycin inHepG2 cells

目的观察雷帕霉素联合应用表阿霉素抑制HepG2细胞的程度，并探讨自噬在其中的作用。方法分组用药后，测定对HepG2细胞增殖的抑制作用；流式细胞仪检测细胞线粒体膜电位，荧光染色法检测自噬体形成以及Westernblot测定p62、Beclinl、人微管相关蛋白1轻链3（LC3）I、LC3lI的蛋白表达水平。结果联合用药后，抑瘤率（67．00±2．45）％较单药组（23．00±1．85）％显著增加（P〈0．05），线粒体膜电位（荧光比61．14％）较单药组（荧光比25．73％）明显下降（P〈0．01），自噬囊泡增多，并明显增加了LC3II／LC3I比值以及p62表达。结论雷帕霉素阻断雷帕霉素靶蛋白（mTOR）通路可诱导HepG2细胞自噬，联合表阿霉素时能增加自噬水平，提高HepG2细胞的化疗敏感性。

Objective To explore the effects of rapamycin combined with epirubicin on prolifera- tion of HepG2 cells and the roles of autophagy. Methods HepG2 cells were given rapamycin combined with epirubiein or alone, and divided into four groups. The inhibitory effects of rapamycin combined with epirubiein or alone on proliferation of HepG2 cells were assessed. Mitoebondrial membrane was tested by u- sing flow cytometry. The autophagic bodies were observed by using fluorescence staining, and the protein expression levels of p62, LC-3 I , LC3 1] and Beclinl were detected by using Western blotting. Results The viability of HepG2 cells in rapamycin ＋ epirubicin group was increased significantly as compared with single group [ （67.00 ±2. 456）% vs. （23.00 ± 1.85）% ,P 〈0.05]. Mitoehondrial membrane in rapam- yein ＋ epirubicin group was decreased significantly as compared with single group [ green fluorescence ratio （61.14%） vs. （25.73%）, P 〈 0. 05 3. In rapamycin ＋ epirubicin group, the expression of p62 and LC3 H was up-regulated. Conclusion Blocking the mammalian target of rapamycin （roTOR） pathway can induce death of cancer cells through autohagie mechanisms, and combined use of rapamyein and epirubiein may synergically enhance the effects of activating autophagy.