上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

细胞自噬对腺花素诱导人肝细胞癌Bel-7402细胞死亡的影响

徐以蔚1，孙汉董2，黄莺1   

  1. 1.上海交通大学 基础医学院病理生理学教研室 细胞分化与凋亡教育部重点实验室， 上海 200025； 2.中国科学院昆明植物研究所 植物化学与西部植物资源持续利用国家重点实验室， 昆明 650201
  • 出版日期:2015-08-28 发布日期:2015-09-30
  • 作者简介:徐以蔚（1989—）， 女， 硕士; 电子信箱: winnie_212@live.com。

Effects of autophagy on adenanthin-induced death of human hepatocellular carcinoma Bel-7402 cells

XU Yi-Wei1, SUN Han-dong2, HUANG Ying1   

  1. 1.Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of the Chinese Ministry of Education, Basic Medicine Faculty of Shanghai Jiao Tong University, Shanghai 200025, China； 2.State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming650201, China
  • Online:2015-08-28 Published:2015-09-30

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摘要：

目的  探讨细胞自噬在腺花素诱导的人肝细胞癌（HCC）Bel-7402细胞死亡中的作用。方法  以9 μmol/L的腺花素处理Bel-7402细胞0、0.5、1、2、4、6、8、12、24 h，Western blotting检测自噬相关蛋白Beclin-1和LC3的蛋白表达。采用免疫荧光方法检测9 μmol/L 腺花素处理0、2、4 h的Bel-7402细胞中内源性LC3蛋白的表达分布情况。通过建立特异性干预Beclin-1基因表达的细胞系，利用Western blotting分析及光学显微镜观察Beclin-1低表达对腺花素诱导的自噬活性改变以及细胞生长抑制和死亡的影响。利用CCK-8方法检测野生型和ATG5缺失（ATG5-/-）小鼠胚胎成纤维细胞（MEFs）对于不同浓度的腺花素诱导的细胞生长抑制效应，通过比较半数抑制浓度（IC50），分析ATG5基因沉默MEFs与野生型MEFs的腺花素敏感性差异。结果  Western blotting和免疫荧光检测结果显示，腺花素显著增强Bel-7402细胞的自噬活性。敲除Beclin-1基因对腺花素诱导的细胞自噬活性增强效应及其引发的细胞死亡过程无明显影响。与野生型MEFs比较，ATG5-/- MEFs对腺花素诱导的细胞生长抑制效应更为抵抗。结论  腺花素能够显著增强Bel-7402细胞的自噬活性，并促进其诱导细胞死亡的发生，但自噬活性的增强并不依赖Beclin-1的表达。

关键词: 腺花素, 自噬, 肝细胞癌, 细胞死亡

Abstract:

Objective  To investigate the effects of autophagy on adenanthin-induced death of human hepatocellular carcinoma (HCC) Bel-7402 cells. Methods  Bel-7402 cells were treated by adenanthin of 9 μmol/L for 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 h. Expressions of autophagy related proteins Beclin-1 and LC3 were detected by Western blotting. The distribution of expression of endogenous LC3 protein in Bel-7402 cells treated by adenanthin of 9 μmol/L for 0, 2, and 4 h was detected by immunofluorescence.The cell line that the expression of Beclin-1 gene was specifically intervened was constructed. The effects of low expression of Beclin-1 on changes of autophagy activity, cell growth, and cell death induced by adenanthin were analyzed and observed by western blotting and light microscopy, respectively.  CCK-8 was adopted to detect the effects of wild type and ATG5 knockout (ATG5-/-) mouse embryonic fibroblasts (MEFs) on the inhibition of cell growth induced by adenanthin of different concentrations. The difference of sensitivity to adenanthin between wild type and ATG5 knock out MEFs was analyzed by comparing the IC50. Results  Western blotting and immunofluorescence showed that adenanthin significantly increased the autophagy activity of Bel-7402 cells. Knockout of Beclin-1 did not remarkably affect adenanthin-induced autophagy activity and cell death. Compared with wild type MEFs, ATG5-/- MEFs were more resistant to the inhibition of cell growth induced by adenanthin. Conclusion Adenanthin can significantly increase the autophagy activity of Bel-7402 cells and promote adenanthin-induced cell death. But the increase of autophagy activity is independent of the expression of Beclin-1.

Key words: adenanthin, autophagy, hepatocellular carcinoma, cell death