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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Beclin1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-Beclin1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24h后收集细胞,分别提取细胞总蛋白,通过Western blot检测Beclin1、Bcl-2和Bax蛋白表达,鉴定转染效率。应用1mmol/LH2O2作用Beclin1-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/AnnexinV-FITC双染色法检测细胞凋亡率。结果显示,Beclin1-siRNA重组质粒明显降低Beclin1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,1mmol/LH2O2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1mmol/LH2O2组相比,转染Beclin1-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-Beclin1转染神经胶质瘤U251细胞后,可有效抑制Beclin1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。
Oxidative stress can induce autophagy and apoptosis simultaneously, but the role of autophagy remains unclear. Studies have shown that Beclin1 is involved in the oxidative stress injury of glioma as a key gene regulating the formation of pre-autophagosomes process. In order to investigate the role of autophagy in H2O2-induced glioma U251 cell injury, we used eukaryotic cell transfection technology to transfer Psilencer3.1-siRNA-Beclin1 recombinant plasmid into human glioma U251 cells, Transfection of empty plasmid negative control group and transfection reagent negative control group. The cells were collected after 24 hours, and the total cellular protein was extracted respectively. The expression of Beclin1, Bcl-2 and Bax proteins were detected by Western blot, and the transfection efficiency was identified. Beclin1-siRNA cell line was treated with 1mmol / L H2O2 and the autophagic vacuoles were detected by monodansylcadaverine (MDC) staining. The expression of autophagy LC3 was detected by Western blot. The flow cytometry PI / Annexin V-FITC Double staining method to detect apoptosis rate. The results showed that Beclin1-siRNA recombinant plasmid significantly reduced the expression of Beclin1 protein and had no significant effect on the expression of Bcl-2 and Bax. Compared with the normal control group, U251 cells treated with 1mmol / LH2O2 exhibited autophagy Compared with 1mmol / LH2O2 group, the expression of LC3-II protein increased and the apoptosis rate increased (P <0.05). Compared with 1mmol / LH2O2 group, the transfection of Beclin1-siRNA plasmid reduced the H2O2- (P <0.05). When combined with 3-methyladenine (3-MA), a specific inhibitor of autophagy, the apoptosis rate of U251 cells was significantly increased (P < 0.05). The above results showed that Psilencer3.1-siRNA-Beclin1 transfected glioma U251 cells can effectively inhibit Beclin1 protein expression, reduce H2O2-induced autophagy levels, increase the rate of apoptosis, and add autophagy inhibitors The 3-MA results are consistent. This study suggests that autophagy is a cell self-protection mechanism in the process of oxidative stress, inhibition of autophagy promotes apoptosis.