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抑制Beclin 1促进H2O2诱导的神经胶质瘤U251细胞凋亡

孔晓霞, 张宏宇*, 陈兆琴, 范小芳, 龚永生

温州医学院1低氧医学研究所,机能实验教学中心;药学院,温州 325035

摘要

氧化应激能够引起细胞自噬和凋亡同时发生,但其中细胞自噬的作用仍不十分明确,研究表明Beclin 1作为调节前自噬体形成的关键基因,参与了胶质瘤氧化应激的损伤过程。为了探讨自噬在H2O2引起的神经胶质瘤U251细胞损伤中的作用,本文应用真核细胞转染技术将Psilencer3.1-siRNA-Beclin 1重组质粒转入人神经胶质瘤U251细胞,同时分别设立转染空质粒阴性对照组和转染试剂阴性对照组。于24 h后收集细胞,分别提取细胞总蛋白,通过Western blot检测Beclin 1、Bcl-2和Bax蛋白表达,鉴定转染效率。应用1 mmol/L H2O2作用Beclin 1-siRNA细胞株,单丹磺酰尸胺(monodansylcadaverine, MDC)染色检测细胞自噬空泡的变化;Western blot检测自噬蛋白LC3表达;流式细胞仪PI/Annexin V-FITC双染色法检测细胞凋亡率。结果显示,Beclin 1-siRNA重组质粒明显降低Beclin 1蛋白表达,并且对凋亡相关蛋白Bcl-2和Bax表达无明显影响;与正常对照组相比,1 mmol/L H2O2作用的神经胶质瘤U251细胞自噬空泡明显集聚,LC3-II蛋白表达增强,细胞凋亡率增加(P < 0.05);与1 mmol/L H2O2组相比,转染Beclin 1-siRNA质粒后降低了H2O2引起的自噬空泡集聚,LC3-II蛋白表达下降,但细胞凋亡率显著增加(P < 0.05);H2O2与自噬特异性抑制剂3-methyladenine (3-MA)联合应用时,U251细胞凋亡率亦显著增加(P < 0.05)。上述结果表明,Psilencer3.1-siRNA-Beclin 1转染神经胶质瘤U251细胞后,可有效抑制Beclin 1的蛋白表达,降低H2O2引起的细胞自噬水平,增加细胞凋亡率,与添加自噬抑制剂3-MA的结果一致。本研究提示自噬在氧化应激过程中是一种细胞的自我保护机制,抑制自噬促进了细胞凋亡的发生。

关键词: RNA干扰; Beclin 1; 自噬; 凋亡

分类号:R73

[Inhibition of Beclin 1 enhances apoptosis by H2O2 in glioma U251 cells.] [Article in Chinese]

KONG Xiao-Xia, ZHANG Hong-Yu*, CHEN Zhao-Qin, FAN Xiao-Fang, GONG Yong-Sheng

Institute of Hypoxia Medical Research, Teaching Center of Functional Experiment; Pharmacy School, Wenzhou Medical College, Wenzhou 325035, China

Abstract

Oxidative stress could induce apoptosis and autophagy process simultaneously, but the role of autophagy is still not clear. Beclin 1, a key gene regulating the preautophagosome formation, is involved in the injury induced by oxidative stress. To observe the role of autophagy in H2O2-induced injury of U251 cells, the recombinant plasmid Psilencer3.1-siRNA-Beclin 1 was transfected into U251 cells by eukaryotic cell transfection technique. Plasmid vector and cell culture medium were used as negative and control groups respectively. The cells were collected 24 h later, and the cell total protein was extracted to detect Beclin 1, Bcl-2 and Bax protein expressions by Western blot. After the Beclin 1-siRNA cells were treated with 1 mmol/L H2O2, the autophagic vacuoles in the cells were stained with monodansylcadaverine (MDC), and the cell apoptotic ratio was determined with PI/Annexin V-FITC staining by flow cytometry analysis. The results showed that the synthetic siRNA decreased the expression of Beclin 1 protein significantly, but had no obvious effect on the levels of Bcl-2 and Bax protein expressions. Compared with those in the control group, the autophagic vacuoles, the level of LC3-II protein expression and the percentage of apoptotic cells increased (P < 0.05) in 1 mmol/L H2O2 group. In Beclin 1- siRNA + H2O2 group, autophagic vacuoles and the levels of LC3-II protein expression decreased obviously, the percentage of apoptot ic cells increased significantly compared with that in 1 mmol/L H2O2 group (P < 0.05). H2O2 and autophagy inhibitor 3-methyladenine (3-MA) combination also increased the percentage of apoptotic cells obviously (P < 0.05). These results revealed that the transfection of Psilencer3.1-siRNA-Beclin 1 effectively inhibited the expression of Beclin 1 protein expression, degraded the autophagy level and increased the apoptotic rate in U251 cells under oxidative stress, which was coincident with the effect of autophagy inhibitor 3-MA. This study suggests that autophagy is a cell protective role in oxidative stress process, and the inhibition of autophagy may enhance apoptosis.

Key words: RNA interference; Beclin 1; autophagy; apoptosis

收稿日期:2010-12-11  录用日期:2011-04-08

通讯作者:张宏宇  E-mail: st.hyz@hotmail.com

引用本文:

孔晓霞, 张宏宇, 陈兆琴, 范小芳, 龚永生. 抑制Beclin 1促进H2O2诱导的神经胶质瘤U251细胞凋亡[J]. 生理学报 2011; 63 (3): 238-244.

KONG Xiao-Xia, ZHANG Hong-Yu, CHEN Zhao-Qin, FAN Xiao-Fang, GONG Yong-Sheng. [Inhibition of Beclin 1 enhances apoptosis by H2O2 in glioma U251 cells.] [Article in Chinese]. Acta Physiol Sin 2011; 63 (3): 238-244 (in Chinese with English abstract).