【作者】 孔晓霞；

【导师】 李扬； 孙连坤；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 目的:探讨氧化应激诱导神经胶质瘤U251细胞自吞噬和凋亡中相关的分子机制以及抗凋亡基因Bcl-2在此过程中的作用,为神经胶质瘤治疗研究提供新的理论思路与实验依据。方法:MTT法、Hoechst 33342染色、RT-PCR和Western Blot等方法检测H₂O₂对细胞增殖、自吞噬及凋亡的影响;以基因重组技术构建pEGFP-C1-Bcl-2质粒;应用真核细胞转染技术,转染人神经胶质瘤U251细胞株,观察过表达Bcl-2对细胞增殖、自吞噬及凋亡方面的影响;通过免疫荧光染色,免疫共沉淀技术,流式细胞术,RT-PCR和Western blot等实验技术,检测细胞损伤中相关信号以及基因表达水平的变化。结果:（1）MTT、Hoechst 33342染色、Western Blot检测表明,H₂O₂能引起U251细胞活力下降,染色质凝集,Bcl-2蛋白表达下降,Bax蛋白表达增强,Bax/Bcl-2比值明显增高,Caspase-3信号通路激活,引起细胞色素c释放。RT-PCR检测表明,H₂O₂能引起U251细胞线粒体动力学失衡。（2）H₂O₂能引起U251细胞自吞噬相关蛋白Beclin 1表达增加,抑制自吞噬上游调控信号Akt、mTOR磷酸化激活。透射电镜结果表明H₂O₂能引起U251细胞空泡化。激光共聚焦显微镜观察免疫荧光染色结果,发现H₂O₂在引起细胞染色质浓缩的同时,也可以引起自吞噬标志物MDC的积聚,以及吖啶橙染色的阳性红色荧光显著增强。（3）通过酶切鉴定、测序、激光共聚焦显微镜检测、RT-PCR和Western Blot等方法鉴定pEGFP-C1-Bcl-2重组质粒以及建立稳定转染细胞系。（4）流式细胞术及RT-PCR检测表明,过表达Bcl-2能够部分抑制细胞内活性氧的产生以及线粒体膜电势下降,稳定线粒体动力学,进而抑制细胞凋亡。（5）通过免疫共沉淀实验发现,过表达Bcl-2导致Bcl-2/Beclin 1复合物增加,抑制Beclin 1信号激活。免疫染色及RT-PCR和Western Blot结果表明过表达Bcl-2可以抑制H₂O₂诱导的Akt、mTOR磷酸化信号降低、自吞噬标志物MDC的积聚、酸性空泡增加,LC3-II蛋白积聚,从而抑制自吞噬的发生。（6）MDC和吖啶橙染色结果表明3-MA能抑制H₂O₂引起的自吞噬,MTT实验,Annexin V-FITC/PI双染色结果表明,3-MA在抑制H₂O₂引起的自吞噬的同时,引起细胞凋亡率显著增加;而Caspase广谱抑制剂Z-VAD-FMK能够抑制H₂O₂引起的凋亡,但是对自吞噬作用不明显。结论:（1）H₂O₂能引起U251细胞包括Bax/Bcl-2、Caspase-3和细胞色素c在内的线粒体信号通路相关的凋亡,引起线粒体动力学失衡。（2）H₂O₂能引起U251细胞Beclin 1蛋白以及自吞噬上游信号Akt、mTOR相关的自吞噬。（3）应用基因重组技术构建pEGFP-C1-Bcl-2质粒并成功建立U251-Bcl-2稳定细胞株。（4）过表达抗凋亡效应分子Bcl-2能够部分抑制活性氧积聚以及线粒体膜电势下降,稳定线粒体。同时Bcl-2通过影响Beclin 1激活以及Akt、mTOR信号水平,抑制H₂O₂引起的自吞噬。（5）利用自吞噬特异性抑制剂3-MA抑制自吞噬能够促进细胞凋亡的发生,表明氧化应激引起的U251细胞自吞噬很可能是细胞的一种自我保护机制。综上所述,我们推测在H₂O₂诱导U251细胞损伤中,细胞通过自吞噬提供能量促进细胞生存,能在一定程度上拮抗氧化应激造成的细胞损伤。因此,自吞噬抑制药物与抗癌药联合应用,很可能成为肿瘤治疗有效的新策略。更多还原

【Abstract】 Gliomas are by far the most common primary brain tumors in adults. Malignant gliomas are resistant to various proapoptotic therapies,such as radiotherapy and conventional chemotherapy,glioma cells are also resistant to the conventional proapoptotic cancer therapeutics. Therefore, effective treatment of malignant gliomas may rely on the development of novel strategies for inducing nonapoptotic cell death,such as autophagic cell death or cell death through mitotic catastrophe,which has been recently described as alternative death pathways.recent reports suggest a role for ROS as signaling molecules in autophagy, high levels of ROS can oxidize cell lipids, proteins and DNA, causing dysfunction of mitochondria, lysosomes and other organelles.Various defense mechanisms can protect cells against oxidative stress,including the degradation and recycling of damaged cell proteins and organelles by autophagy.H2O2 is active oxygen and intermediate product of oxidation in vivo.it is easy to penetrate cytoplasmic membrane and product cellular toxicity.Atg4,an essential protease in the autophagic pathway, has been identified as a direct target for oxidation by H2O2. However, these pathways remain to be elucidated. In this study, we have demonstrated that H2O2 induces apoptosis and autophagy in U251 glioma cells and construct Bcl-2 Overexpression and Bcl-2 gene-specific small interfering RNA（siRNA）to observe and approach that the role and mechanism of Bcl-2 in H2O2 induces apoptosis and autophagy ,meanwhile discuss the relation about apoptosis and autophagy. And their significance in therapy cancer.A variety of physiological death signals, as well as pathological cellular insults, trigger the genetically programmed pathway of apoptosis.in mitochondria pathway of apoptosis. A major checkpoint in the common portion of this pathway is the ratio of pro-apoptotic（BAX） to anti-apoptotic（BCL-2） members.Downstream of this checkpoint are two major execution programs: the caspase pathway and mitochondria dysfunction. Mitochondrial dysfunction includes a change in the mitochondrial membrane potential , production of reactive oxygen species（ROS）, opening of the permeability transition pore（PTP）,and the release of the intermembrane space protein,cytochrome c（Cyt c）. Released cytochrome c activates Apaf-1, which in turnactivates a downstream caspase program.Apoptosis and autophagy are both tightly regulated biological processes that play a central role in tissue homeostasis, development,and disease.The antiapoptotic protein,Bcl-2,interacts with the evolutionarily conserved autophagy protein, Beclin 1. However, little is known about the functional significance of this interaction. Autophagy is an evolutionarily conserved pathway that involves the sequestration and delivery of cytoplasmic material to the lysosome, where it is degraded and recycled. In theory, autophagy may help promote cell survival,either by purging the cell of damaged organelles, toxic metabolites, and intracellular pathogens or by generating the intracellular building blocks required to maintain vital functions during nutrient-limiting conditions. However, in theory,autophagy may also promote cell death through excessive self-digestion and degradation of essential cellular constituents. Despite recent advances in understanding its molecular mechanisms and biological functions, it is unclear whether autophagy acts fundamentally as a cell survival or cell death pathway—or both.The mechanisms responsible for autophagy are still not very clear.The interaction between the antiapoptotic protein, Bcl-2,and the autophagy protein, Beclin 1, represents a potentially important point of convergence of the apoptotic and autophagic machinery. Beclin 1,the mammalian ortholog of yeast Atg6/Vps30, was first identified in a yeast two-hybrid screen as a Bcl-2-interacting protein and is a haplo insufficient tumor-suppressor gene that is frequently monoallelically deleted in human sporadic breast, ovarian, and prostate cancer . Beclin 1 is part of a Class III PI3K complex that participates in autophagosome formation, mediating the localization of other autophagy proteins to the preautophagosomal membrane.The signaling pathway composed of phosphatidylinositol 3-kinase （PI3K）, protein kinase B （Akt）, and mammalian target of rapamycin （mTOR） plays a central role in the regulation of cell proliferation, differentiation, and survival.The class I PI3K /Akt pathway which is constitutively activated in glioma cells,is known to play an important role in cell survival （inhibition of apoptosis） and has been linked to various human cancers. Disruption of the PI3K/Akt pathway, culminating in inhibition of Akt, has been found to be associated with autophagy induced by a variety of anti-neoplastic agents in cancer cells. Studies have indicated that Bcl-2 can be a strict mediator downstream of PI3K/Akt signaling, and that Akt contributes to the positive regulation of mTOR signaling pathway, which can inhibit cell autophagic activity.In HT-29 cells, the inhibitory effects of Bcl-2 are associated with disruption of the Beclin 1/hVps34 complexes, which are thought to be essential for early stages of autophagosomal formation Upon the induction of autophagy, phosphatidylethanolamine is covalently linked to the cytosolic protein LC3-I to yield LC3-II, which then associates with the autophagosome. This conversion is commonly used as a marker for autophagy .The purpose of this study was to determine whether H₂O₂ can induce programmed cell death in human glioma U251 cells to example the relationship between autophagy and apoptosis in this process, and to identify the signaling pathways that might be involved.Methods:（1） Cell culture（2） The Bcl-2 cDNA was amplified by PCR and then construct Anti-apoptosis protern Bcl-2 Overexpression vectors and detect their Overexpression and silencing effects by RT-PCR、Western Blot. （3） To detect the mechanisms of H₂O₂-induced glioma U251 cells injured. Expression of Bax、Bcl-2、cytC and Caspase-3 mRNA and protern detect by RT-PCR and Western Blot.and also detect expression of Beclin 1、Akt、FKHR、mTOR、p70S6K、LC 3 protern.（4） MDC, Hoechst 33342 and acridine orange staining were used to detect the autophagic vacuoles and cell apoptotic chromatin condensation by Confocal microscopy。autophagic vacuoles and cell apoptotic chromatin condensation in U251 cells treated with H₂O₂ by transmission electron microscopy.（5） To detect cell vitality and Expression of Mfn1, Mfn2 ,Opa1mRNA of fusion gene and Expression of Fis1, Drp1,MTP18 mRNA of fision gene by MTT and RT-PCR. Expression of Beclin 1、Akt、FKHR、mTOR、p70S6K、LC 3 protein by Western blot. The loss ofΔΨm was measured with Rhodamine123. autophagic vacuoles and cell apoptotic chromatin condensation were detected by Confocal microscopy.The generation of ROS was measured with the ROS-detecting fluorescent dye DCFH-DA.（6） To detect relationship to apoptosis and autophagic cell death though 3-MA and Z-VAD were used in H₂O₂-induced glial cells by Flowcytometry analysis and Immunofluorescent.Results:（1） MTT、Hoechst 33342 dyeing、western blot indicated H₂O₂ induced dose-dependent cell death in malignant glioma U251 cells,Cell vitality decrease, chromatic agglutination。the Bcl-2 proteins expression decreased significantly. the ratio of Bax/Bcl-2 mRNA and protein increased obviously. Caspase-3 signal activation and cytC release.RT-PCR result indicated H₂O₂ induced disruption of mitochondrial dynamics.（2） At the same time to detect the autophagy induced by H₂O₂, we have found The expression of autophagy-associated Beclin 1 protein was up-regulated and significant reduction of Akt, FKHR, mTOR and p70S6K phosphorylation. MDC staining can be used to detect autophagic vacuoles. compared with the control, autophagosomes were increased in U251 cells after exposed to H₂O₂ treatment.AO stain indicated red fluorescence increase. （3） We have constructed successfully a recombinant Overexpression plasmid of Bcl-2 ,and have also confirmed by restrictive enzyme digestion and DNA sequencing. RT-PCR and Western Blot analysis revealed a strongly decreased level of Bcl-2 mRNA, And Bcl-2 mRNA and protern do increased strongly in U251 cells transfected with the pEGFP-C1-Bcl-2 compared with the negative control.（4） flow cytometry and RT-PCR result indicated overexpression of Bcl-2 inhibited Cell vitality decrease and the generation of ROS and also inhibition the loss ofΔΨm induced by H₂O₂.（5） Co-immunoprecipitation of Beclin 1 and Bcl-2 in non-transfected U251 cells and U251 cells transfected with the overexpressed Bcl-2 vector indicate Bcl-2 co-immunoprecipitated with more endogenous Beclin 1 than in control U251 cells inhibiting autophagy though the class III PI3K/Beclin 1 pathway .on other hand, Bcl-2 inhibiting autophagy though decreased Akt、mTOR proteins expression. MDC staining indicated cell acidification and formation of autophagic vacuoles.（6） MTT assay indicated that 3-MA or Z-VAD-FMK alone had no effect on cell viability.MDC and AO staining indicated 3-MA treatment enhanced the autophagy induced by H₂O₂. MTT and Annexin V-FITC/PI staining indicated Inhibition autophagy by 3-MA accelerates the apoptosis induced by H₂O₂. the addition of Z-VAD-FMK increased cell viability of H₂O₂ treated cell but viability was still decreased compared with the control group,but the addition of Z-VAD-FMK have no effect with autophagy.Conclusions:（1） H₂O₂ can induced glioma U251 cells apoptosis, and mitochondria signal include Bax/Bcl-2、Caspase-3 and cytC participated in the regulation of apoptosis. H₂O₂ induces loss of mitochondrial membrane potential and disruption of mitochondrial dynamics.（2） H₂O₂ can induced glioma U251 cells autophagy though both Beclin 1 and Akt/mTOR signaling.（3） we have identified that successfully constructed a recombinant overexpression Bcl-2 plasmid and also successfully constructed U251-Bcl-2 stable cells.（4） Bcl-2 can partial inhibited ROS accumulation and decrease mitochondria membrane potential stablized mitochondria.at the same time, Bcl-2 can inhibited autophagy induced by H₂O₂ though both Beclin 1 and Akt/mTOR signaling.（5） Inhibition autophagy by 3-MA accelerates the apoptosis induced by H₂O₂.we presume autophagy induced by H₂O₂ in glioma U251 cells could protect cells from injure.In glioma U251 cells injure induced by H₂O₂, autophagy can portect cells though supplying energy, and rivalry oxidative stress injure, thereby infer that autophagy suppressant and anticancer drugs combination application may become new and effective strategy to tumor therapy.更多还原