细胞自噬在三氧化二砷诱导肝癌细胞凋亡中的作用研究

【摘要】目的研究细胞自噬在三氧化二砷（ATO）诱导的肝癌细胞死亡中的作用，观察调控细胞自噬对ATO诱导的细胞凋亡有何影响。方法以人肝癌HepG2细胞为研究对象，在ATO作用于HepG2细胞的基础上，加入自噬激动剂〔雷帕霉素（Rap）〕和自噬抑制剂〔三甲基腺嘌呤（3-MA）〕进行调控。以MTT法检测细胞生存率，透射电镜观察自噬体结构，免疫印迹法分析自噬相关蛋白LC-3和Beclin1的表达；流式细胞术检测细胞凋亡。结果 5~20 μmol/L ATO与10 mmol/L 3-MA联用时HepG2细胞存活率比同剂量ATO单独作用时降低（P <0.05），凋亡率增加（P <0.05），细胞内呈现典型凋亡反应特征，自噬蛋白LC3和Beclin1表达低于ATO单独作用组（P <0.05）。相反，5~20 μmol/L ATO与0.25 μmol/L Rap联用时，HepG2细胞的存活率比ATO单独作用时显著增加（P <0.05），凋亡率降低（P <0.05），细胞内出现大量自噬体结构，自噬蛋白LC3和Beclin1的表达较ATO单独作用组有增加趋势，但差异无统计学意义（P >0.05）。结论特异性自噬抑制剂能显著增强ATO杀伤肝癌细胞的敏感性，其机制与增强肝癌细胞凋亡有关。

英文摘要:

【Abstract】 Objective To determine the effect of autophagy on the apoptosis of hepatocellular carcinoma cells induced by arsenic trioxide (ATO). Methods Hepatocellular carcinoma HepG2 cells were exposed to ATO. The cell viability was detected by MTT after adjustments for autophagy agonist (Rap) and autophagy inhibitor (3-MA). The autophagosome was observed under electronic microscope. The autophagy related proteins (LC3 and Beclin1) were detected by immunofluorescence. The cell apoptosis was measured by flow cytometry. Results With 5-_{20 μmol/L}of ATO, HepG2 cells exposed to 3-MA had significantly lower viability (P <0.05) and higher early apoptosis (P <0.05) than those without exposure to 3-MA. Exposure to 3-MA was also associated with lower expressions of LC3 and Beclin1, with HepG2 cells showing typical apoptotic characteristics. By contrast, with 5-_{20 μmol/L}of ATO, the cells exposed to Rap showed significantly higher viability (P <0.05) and lower early apoptosis (P <0.05) than those without exposure to Rap. A large number of autophagosome appeared in the cells exposed to Rap. Exposure to Rap was associated with increased expressions of LC3 and Beclin1, but with no statistical significance (P >0.05). Conclusion Targeted autophagy inhibition can significantly increase the sensitivity of HepG2 to ATO. The underlining mechanism is associated with enhanced apoptosis of hepatocellular carcinoma cells.

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