JNK在氧化应激导致胶质瘤细胞Parthanatos中的作用及机制研究

发布时间：2018-04-18 12:43

本文选题：JNK + ROS　；参考：《吉林大学》2016年博士论文

【摘要】：背景:胶质瘤是最常见的一种原发恶性脑肿瘤,发病率较高[25,26]。胶质瘤细胞经放、化疗后仍能存活是因为细胞抗凋亡[26]。PARP-1,是一种高表达的核蛋白,参与检测DNA链断裂,在神经胶质瘤细胞基因组的稳定中参与DNA修复[27],PARP-1轻度激活被认为是胶质瘤细胞抵抗凋亡的一个因素,而抑制PARP-1活化是恢复胶质瘤细胞对化疗或放疗敏感的一种策略[28-31]。最新报道表明,PARP-1过度活化导致细胞死亡[32,33],这是一种新的程序性细胞死亡方式,命名为“Parthanatos”以区别caspase依赖的细胞凋亡、坏死和其他形式的细胞死亡[27]。根据细胞死亡命名委员会的标准,Parthanatos的定义有两个主要准则:一是PAR过度合成伴随细胞死亡,二是细胞死亡因PARP-1基因敲除或PARP-1抑制剂处理后完全或部分抑制[34]。实验研究表明,Parthanatos不仅涉及多种病理状态,如糖尿病、炎症、脑缺氧/缺血和脑外伤[34-37],而且参与化学药物诱导的癌细胞死亡如神经胶质瘤、食管鳞状细胞癌和胃癌[32,33,38]。作为一个独特的细胞死亡途径,Parthanatos的特征包括一系列精确运行的生化反应如PARP-1过度激活,胞浆PAR聚合物积聚,线粒体去极化和AIF核转位。最后,AIF启动核染色质溶解或冷凝导致细胞死亡[27,34]。基因毒性药物诱导DNA链断裂激活PARP-1[27],ROS也参与调节Parthanatos。Chiu等人证明,MNNG(N-甲基-N-硝基-N-亚硝基胍)作用于小鼠胚胎成纤维细胞能抑制ROS水平,减少PARP-1-依赖的细胞死亡。MNNG是一种烷化剂,可以直接损伤DNA[39]。在我们以前的研究中也发现ROS导致脱氧鬼臼毒素作用的胶质瘤细胞PARP-1过度活化[32]。然而氧化应激条件下胶质瘤细胞中PARP-1过度激活涉及的信号通路仍不清楚,有报道MAPK家族的ERK通路参与调控人类淋巴细胞氧化应激引起的Parthanatos[40]。JNK,是MAPK的另一个亚家族,也可以被ROS激活调节胶质瘤细胞程序性死亡[41]。因此,我们推测JNK通过氧化应激调节Parthanatos。H_2O_2、超氧自由基和羟基自由基均属于ROS[16],广泛用于诱导多种癌细胞如胃癌,宫颈癌,大肠癌,乳腺癌和胶质瘤的氧化应激反应[17-24],因此我们用人脑胶质瘤细胞株和H_2O_2探讨JNK在氧化应激导致胶质瘤细胞Parthanatos中的作用。目的:使用胶质瘤细胞系和H_2O_2探讨JNK怎样调节氧化应激导致的胶质瘤细胞Parthanatos方法:1、通过MTT法检测H_2O_2作用的胶质瘤细胞生存活性。2、通过LDH释放实验检测H_2O_2作用的胶质瘤细胞死亡情况。3、通过Annexin V-FITC/PI双染和罗丹明染色流式细胞术检测胶质瘤细胞存活率和线粒体膜电位变化。4、通过DCFH-DA探针检测细胞内ROS水平,Mito SOX探针检测线粒体过氧化物,荧光显微镜观察细胞荧光亮度,荧光密度仪分析荧光密度变化。5、通过免疫细胞化学染色观察H_2O_2作用的胶质瘤细胞内AIF的变化。6、通过JNK Si RNA或PARP-1 Si RNA转染明确JNK、PARP-1在H_2O_2诱导胶质瘤细胞Parthanatos中的作用。7、通过免疫蛋白印迹检测H_2O_2作用的胶质瘤细胞PAR、PARP-1、JNK、p-JNK、AIF的表达变化。结果:1、H_2O_2导致SHG-44,U251和U87胶质瘤细胞生存活性下降;H_2O_2浓度和时间依赖引起SHG-44、U251和U87胶质瘤细胞死亡。3AB抑制PARP-1、Si RNA敲除PARP-1、NAC抑制细胞内ROS或SP600125抑制JNK磷酸化显著提高胶质瘤细胞的生存活性。2、流式细胞术检测显示3AB抑制PARP-1提高胶质瘤细胞的存活率,3AB抑制PARP-1阻止胶质瘤细胞线粒体膜电位下降,Si RNA敲除PARP-1、NAC抑制ROS或SP600125抑制JNK磷酸化提高胶质瘤细胞存活率和减少细胞死亡。3、DCFH-DA探针和Mito SOX探针检测H_2O_2作用的胶质瘤细胞具有更明亮的绿色荧光和红色荧光(对荧光敏感)。荧光密度统计分析证明外源性H_2O_2诱导细胞内ROS和线粒体过氧化物产生、NAC抑制ROS或SP600125抑制JNK磷酸化减少细胞内ROS积聚、SP600125抑制JNK磷酸化减少线粒体过氧化物产生。4、免疫细胞化学染色观察H_2O_2作用的胶质瘤细胞AIF明显积聚于胞核。5、免疫蛋白印迹法检测胶质瘤细胞PAR、PARP-1、JNK、p-JNK、AIF的表达变化:H_2O_2导致胶质瘤细胞PARP-1在胞浆、胞核表达上调,胞浆PAR聚合物形成、胞核内AIF增加;3AB抑制PARP-1、Si RNA敲除PARP-1或NAC抑制ROS,抑制PARP-1的表达、胞浆PAR聚合物形成和AIF核转位。H_2O_2导致胶质瘤细胞JNK磷酸化增加;SP600125抑制JNK或Si RNA敲除JNK抑制JNK磷酸化同时PARP-1表达下降、胞浆PAR聚合物和AIF核转位减少。结论:1、ROS诱导胶质瘤细胞Parthanatos。2、JNK激活促进胶质瘤细胞Parthanatos。3、JNK通过调节细胞内ROS水平参与调节胶质瘤细胞Parthanatos。
[Abstract]:Backgroundglioma is one of the most common primary malignant brain tumors, with high incidence of [25,26]. glioma cells after radiotherapy, chemotherapy survived because of anti apoptotic [26].PARP-1 cells, is a high expression of nuclear protein involved in the detection of DNA strand breaks, involved in DNA repair of [27] in glioma cell genome stable, mild PARP-1 activation is considered to be a factor of glioma cell resistance to apoptosis, and inhibition of PARP-1 activation is the recovery of glioma cells to chemotherapy or radiation sensitive strategy for the new [28-31]. report showed that PARP-1 activation induced cell death of [32,33], which is a new kind of programmed cell death, named the apoptosis of Parthanatos cells to differentiate caspase dependent, necrosis and other forms of cell death in [27]. cell death according to standard nomenclature committee, the definition of Parthanatos has two main criteria: PAR is synthesized with excessive cell death, cell death was two for PARP-1 gene knockout or PARP-1 inhibitor treatment after complete or partial inhibition of [34]. showed that Parthanatos is not only involved in a variety of pathological conditions, such as diabetes, inflammation, brain injury and brain ischemia / hypoxia [34-37], and is involved in chemical drugs induced cancer cell death such as glial tumor, esophageal squamous cell carcinoma and gastric cancer [32,33,38]. as a unique cell death pathway, Parthanatos features include a series of biochemical reactions such as the accurate operation of the excessive activation of PARP-1, cytosolic PAR polymer accumulation, mitochondrial depolarization and nuclear translocation of AIF. Finally, AIF start the nuclear chromatin condensation or dissolution leads to cell death [27,34]. genotoxic drugs induced DNA strand breaks activate PARP-1[27] and ROS are also involved in the regulation of Parthanatos.Chiu proved that, MNNG (N- methyl -N- nitro -N- nitroso guanidine) for For mouse embryonic fibroblast cells can inhibit the level of ROS, reduce the PARP-1- dependent cell death.MNNG is an alkylating agent that can damage DNA[39]. directly in our previous studies also found that ROS in PARP-1 glioma cells Deoxypodophyllo toxin effect but the excessive activation of [32]. under oxidative stress in PARP-1 glioma cells activated signal pathways involved remain unclear, there are reports of MAPK family ERK pathway involved in human lymphocyte oxidative stress induced by regulation Parthanatos[40].JNK, is another subfamily MAPK, can also be activated by ROS regulating programmed cell death in glioma cell lines [41]. therefore, we speculate that JNK Parthanatos.H_2O_2 regulated by oxidative stress, superoxide radicals and hydroxyl radicals base are belong to ROS[16], widely used to induce various cancer cells such as gastric cancer, cervical cancer, colorectal cancer, breast cancer and glioma of oxygen should be Shock response [17-24], so we used the human glioma cell line H_2O_2 and to explore the effect of JNK on Parthanatos glioma cells in oxidative stress. Objective: using glioma cell lines and explore how to adjust the H_2O_2 JNK glioma cell Parthanatos caused by oxidative stress: 1, by glioma cell survival activity detection of H_2O_2 MTT.2 the death of.3 releasing assay of H_2O_2 by LDH glioma cells, the detection of glioma cell survival rate and mitochondrial membrane potential changes of.4 flow cytometry by Annexin V-FITC/PI double staining and Luo Danming staining, the level of ROS DCFH-DA probe detection cells, detection of mitochondrial peroxide Mito SOX probe, cell fluorescence brightness observation fluorescence microscope, fluorescence analysis of density change of.5 fluorescence density meter, glioma cells we observe the effect of H_2O_2 in AIF by immunocytochemical staining The changes of.6, Si RNA or PARP-1 Si JNK by RNA JNK PARP-1 in H_2O_2 transfected with clear, induced by.7 glioma cells in Parthanatos glioma cells, by PAR, Western blot detection of H_2O_2 PARP-1 JNK, p-JNK function, and the expression of AIF. Results: 1, H_2O_2 lead to SHG-44, and decreased U251 U87 glioma cell survival activity; H_2O_2 concentration and time dependent caused by SHG-44, U251 and U87 glioma cell death inhibition of.3AB PARP-1, Si RNA knockdown of PARP-1 NAC inhibition of intracellular ROS or SP600125 inhibited JNK phosphorylation significantly improve the survival activity of.2 glioma cells, flow cytometry showed that 3AB inhibited PARP-1 to improve survival the rate of glioma cells, 3AB glioma cells inhibition of PARP-1 prevented mitochondrial membrane potential decrease, Si RNA knockdown of PARP-1 inhibited ROS, NAC or SP600125 inhibited the phosphorylation of JNK increased glioma cell survival rate and reduce cell death.3, DC Detection of H_2O_2 glioma cells FH-DA probe and Mito SOX probe has a bright red and green fluorescence (on sensitive fluorescence). The fluorescence density statistical analysis proved that exogenous H_2O_2 induced by ROS and mitochondrial superoxide generation in cells, NAC inhibited ROS or SP600125 inhibited JNK phosphorylation reduced intracellular ROS accumulation, SP600125 inhibition of JNK decreased phosphorylation of mitochondrial superoxide production.4, immunocytochemical staining of AIF glioma cells to observe the role of H_2O_2 was accumulated in the nuclei of.5, detection of glioma PAR cells by Western blot, PARP-1, JNK, p-JNK, the expression of AIF: H_2O_2 to PARP-1 glioma cells in the cytoplasm, nucleus expression cell plasma PAR polymer formation, nucleus AIF increased; 3AB inhibited PARP-1, Si RNA or NAC knockdown of PARP-1 inhibited ROS, inhibiting the expression of PARP-1, cytosolic PAR polymer formation and AIF.H_2O_2 induced nuclear translocation Increased phosphorylation of JNK glioma cells; inhibition of SP600125 JNK or Si RNA knockdown of JNK inhibited JNK phosphorylation and decreased expression of PARP-1, cytosolic PAR polymer and AIF nuclear translocation decreased. Conclusion: 1. ROS glioma cells induced by Parthanatos.2, JNK activation promotes glioma cells Parthanatos.3, JNK by regulating the intracellular level of ROS involved in the regulation of Parthanatos. glioma cells

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.41

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