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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).