【作者】 季光；

【导师】 胡治平；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 研究背景及目的：氧化应激(Oxidative stress)是活性氧、活性氮等自由基显著增加,超出内源性抗氧化系统的清除能力时,造成细胞或组织损伤的一种病理反应。氧化应激被认为是多种疾病发生、发展及导致机体衰老的重要因素,其在神经系统疾病如神经退行性病变、脑卒中等中的作用也日益受到重视。高尔基体(Golgi apparatus, GA)是细胞新陈代谢的重要细胞器,越来越多的证据表明,高尔基体在氧化应激中参与转导应激信号,触发凋亡。并且,高尔基体在多种氧化应激介导的病理生理反应及氧化应激作为重要发病机制的神经系统疾病中表现出多种多样的形态变化,其中,高尔基体碎裂(Golgi fragmentation)是神经退行性疾病中观察到的一种典型表现,甚至被认为是神经元凋亡的始动因素。因此,高尔基体形态变化很可能在未来成为神经变性的重要病理指标,具有重要研究价值。高尔基体重组和堆叠蛋白(Golgi reassembly and stacking proteins, GRASPs)是高尔基体囊膜结构的重要组分,它们在高尔基体组装、细胞迁移、细胞分裂、细胞凋亡、蛋白质运输等多种细胞功能中发挥重要作用。GRASPs包括GRASP65及GRASP55。其中,主要位于顺面高尔基体(cis-Golgi network, CGN)的GRASP65被研究较多。研究发现,GRASP65参与了多个生理及病理生理过程中的高尔基体形态改变,如细胞迁移中的高尔基体和中心体定向,细胞分裂期和细胞分裂间期高尔基体解聚、裂解以及细胞凋亡中的高尔基体碎裂。细胞外信号调节激酶(extracellular signal-regulated kinase, ERK)是丝裂原活化蛋白激酶(mitogen-activated protein kinases, MAPKs)系统中的重要成员,参与了包括细胞增殖、存活、凋亡、迁移等在内的多种细胞功能。大量证据表明MAPK/ERK通路在氧化应激中被激活,并参与介导了氧化应激所致的神经细胞损伤。同时,已有研究发现ERK通路在细胞有丝分裂间期通过磷酸化高尔基体结构蛋白GRASP65,介导高尔基体解聚、裂解。由于高尔基体及其形态变化在氧化应激中占有重要地位,而ERK通路在氧化应激中对高尔基体及其相关蛋白作用的研究相对较少,本研究将以小鼠神经瘤N2a细胞为研究对象,以H2O2诱导氧化应激反应,探讨ERK通路在氧化应激中对GRASP65及高尔基体形态变化的影响。方法：1.氧化应激模型的建立：以0、60、70、80、90、100μM H2O2处理N2a细胞,分别在Oh,1h,3h,6h,12h,24h以MTT法检测N2a细胞的增殖抑制率,选择适合的作用浓度并观察H202作用下N2a形态变化；2.MTT法检测MEK/ERK抑制剂U0126对H2O2作用下N2a细胞存活率的影响,选择适宜的H2O2作用时间；3.实验分为正常对照组、H2O2损伤组和U0126抑制组,其中H2O2损伤组和U0126抑制组分别设置1h,3h,6h亚组；4. Western blot技术检测各组中ERK,pERK及GRASP65, pGRASP65的表达；5.免疫荧光激光共聚焦观察GRASP65、pGRASP65的表达及高尔基体形态变化；6.流式双染法检测各组细胞凋亡率。结果：1.MTT检测结果显示,H2O2对N2a细胞具有损伤作用,且这种损伤作用具有浓度和时间依赖性,浓度越大,时间越长,对细胞的损伤作用越强；H2O2作用下,随着时间延长,N2a细胞发生明显变化。2.随时间延长,H2O2损伤组与U0126抑制组N2a细胞存活率均逐渐下降。与H2O2损伤组相比,U0126抑制组在1h,3h提高了N2a细胞的存活率(P<0.01),在6h,12h,24h药物干预组与H2O2损伤组N2a细胞存活率无显著性差异(P>0.05)。3. Western blot检测ERK在各组中的活化：结果显示,ERK在各组中表达无明显变化。pERK的表达在各实验组中出现了明显变化：H202作用1h, pERK表达即出现明显升高,较正常对照组有显著差异(P<0.01); H2O2作用3h, pERK表达水平达到峰值(P<0.01)；H202作用6h, pERK表达仍处于较高水平(P<0.01),但较前开始下降。U0126干预后,pERK在各时间点表达均较H202损伤组降低(P<0.01),表明U0126干预成功抑制了ERK在氧化应激中的活化。4. Western blot检测U0126对GRASP65及pGRASP65表达影响：结果显示,各实验组中GRASP65表达与正常对照组无明显差异(P>0.05)。而在H202损伤组中,pGRASP65在各时间点表达均较正常对照组有显著升高(P<0.01),表明GRASP65在氧化应激中发生了磷酸化。U0126干预后,pGRASP65表达均较同时间点H202损伤组显著降低(P<0.01),提示阻断ERK活化抑制了氧化应激中GRASP65的磷酸化。5.免疫荧光共聚焦结果显示,正常对照组中,GRASP65标记的高尔基体结构紧凑,呈新月形紧密围绕于位于细胞核周围；pGRASP65未见明显表达。在H2O2损伤组中,pGRASP65表达明显增强,且与GRASP65表达区域重合；H202损伤6小时后高尔基体表现出结构松散,呈不规则形态改变；U0126干预后,GRASP65表达较H202损伤组无明显变化,pGRASP65在各时间点表达较H202损伤组明显减弱,其表达区域与GRASP65重合,在6h亚组高尔基体表现出不规则形态改变,与同时间点H202损伤组相似,并且,U0126抑制组发生高尔基体形态变化的细胞比例与H2O2损伤组无明显差别(P>0.05)。6.流式双染法检测结果显示,H202损伤组与U0126抑制组在各时间点未检测到明显早期细胞凋亡(凋亡率<0.1%),各组与对照组相比无显著性差异(P=NS)。而各组细胞死亡率(晚期凋亡/坏死细胞)与对照组相比均有显著性差异(P<0.01),随着H202作用时间延长,H202损伤组与U0126抑制组细胞死亡率均逐渐增加；并且,在U0126抑制组在各时间点细胞死亡率均较H2O2损伤组减少(P<0.05)。结果表明抑制ERK活化减少了氧化应激中N2a细胞的死亡。结论：1.ERK通路在氧化应激中被激活,并参与介导了氧化应激中的N2a细胞损伤,阻断ERK的活化对这种损伤具有保护作用；2.氧化应激中GRASP65在ERK的作用下发生磷酸化；3.氧化应激中高尔基体发生不规则形态改变,但这一形态变化与ERK介导的GRASP65磷酸化及细胞死亡无关联。更多还原

【Abstract】 Background and Objective:Oxidative Stress is a pathophysiological reaction originated from a significant increase in free radicals, such as reactive nitrogen and reactive oxygen species, which can finally result in cells or tissue injury. Oxidative stress is considered to be responsible for the development of a variety of diseases and the body aging. In recent years, increasing attentions has been paid to its role in neurological diseases such as neurodegenerative diseases, stroke, etc. Golgi apparatus is an important organelle functioning in cell metabolism. More and more evidences indicate that the Golgi apparatus is involved in oxidative stress, assisting in the stress signal transduction. In the meanwhile, the Golgi apparatus also shows a variety of morphological alterations in many physiological and pathophysiological processes and neurodegenerative diseases, mechanisms in which oxidative stress is strongly involved. Furthermore, Golgi fragmentation has been found to be classical feature in neurodegenerative diseases, and is even considered to be an early causative step in neural apoptosis. Since that, neuronal GA morphology may be a reliable index of activity of degeneration and thus should be studied further. Golgi reassembly and stacking proteins (GRASPs) are important components of the Golgi vesicle membranes, which are involved in many cell behaviors such as Golgi stacking, cell migration, cell division, apoptosis, and cargo secretion. GRASPs consist of two proteins, termed GRASP65and GRASP55. GRASP65, which is mainly located at the cis-Golgi, has been well studied and is found to be of great significance in Golgi morphological alterations in a variety of physiological and pathological physiological processes, such as the Golgi and centrosome orientation in cell migration, Golgi deoligomerization and disassembly in mitosis, as well as Golgi fragmentation in cell apoptosis.Extracellular signal-regulated kinase (ERK) is an important member in mitogen-activated protein kinase (MAPKs) system, which is involved in a variety of cellular functions such as cell proliferation, cell survival, cell apoptosis and cell migration. Considerable evidence has revealed that the MAPK/ERK pathway is activated in oxidative stress and contributes to the neuronal damage induced by oxidative stress. At the same time, studies have found that the phosphorylatioon of GRASP65by ERK is required for Golgi disassembly at interphase. Little attention, however, has been paid to the role of ERK pathway on Golgi apparatus and Golgi associated proteins in oxidative stress, in which Golgi apparatus is strongly involved. In this study, we will use the mice neuroma N2a cells in H2O2-induced oxidative stress, exploring the effects of MAPK/ERK pathway on GRASP65and Golgi morphology in oxidative stress.Methods:1. Establish a H2O2-induced oxidative stress model in vitro. The neuronal injury was measured by the MTT assay. Select the appropriate concentration of H2O2according to the inhibition rate, and observe the cell morphological changes under Light Microscopes.2. MTT assay to detect the effect of U0126on cell viability in H2O2-induced oxidative stress. Select the appropriate time for H2O2.3. In order to evaluate the role of ERK in oxidative stress, U0126was used to inhibit the ERK activation. Experimental grouping was designed as follows:control group; H2O2injury group; U0126inhibited group. The latter two groups were further divided into three different subsets for various exposed H2O2injury time (lh,3h,6h).4. Western blot to detect the expression of ERK, pERK and GRASP65, pGRASP65.5. Immunofluorescence laser scanning confocal to observe the morphological alterations of Golgi apparatus, as well as the expression of GRASP65and pGRASP65.6. The rate of cell apoptosis was measured by flow cytometric analysis.Results:1. MTT test showed the H2O2-induced N2a cell injury is concentration-and time-dependent, the higher of the H2O2concentration and the longer of the injury time, the stronger of the damage effect to cells; N2a cells exhibited obvious morphologic changes in H2O2-induced oxidative stress.2. Cell viability in H2O2injury group and U0126inhibited group both decreased with a time-dependent manner. U0126obviously promoted the cell survival in lh,3h compared with that in H2O2injury group (P<0.01); While cell viability in6h,12h,24h between U0126inhibited group and H2O2injury group has no significant difference (P>0.05). These results showed that the inhibition of ERK activation has a protective effect on oxidative stress mediated cell damage not more than6h.3. Western Blot results showed that expression of ERK has no significant difference between that in control group and in experimental groups. Meanwhile, expression of pERK significantly increased (P<0.01) at each time point in H2O2injury group compared with the control group, indicating that ERK activation occured in oxidative stress, and the pERK expression reaches peak at3h in H2O2injury group; U0126intervention reduced the pERK expression at each time point compared with that in H2O2injury group (P<0.01), showing that U0126intervention succesfully supressed the ERK activation in oxidative stress.4. Expression of GRASP65has no significant difference between that in control group and in experimental groups (P>0.05).5. Expression of pGRASP65significantly increased at each time point in H2O2injury group, compared with that in control group (P<0.01), indicating that GRASP65was phosphorylated in oxidative stress, and expression of pGRASP65shared a same trend with that of pERK; After the intervention of U0126, expression of pGRASP65significantly decreased (P<0.01) compared with that in H2O2injury group, indicating that the supression of ERK activation inhibited GRASP65phosphorylation.6. Immunofluorescence confocal:In control group, GRASP65labeled Golgi apparatus is compact, crescent-shaped, locating at the perinuclear region, pGRASP65has no significant expression. In H2O2injury group, expressioon of GRASP65showed no significant difference with that in control group; while pGRASP65expression was significantly increased, and the expression region coincide with that of GRASP65; Golgi apparatus exhibited scattered structure and irregular morphological changes at6h; In U0126inhibited group, there is no significant changes of GRASP65expression than that in H2O2injury group, and pGRASP65expression was significantly weakened at each time points compared with H2O2injury group, which share the same expression region to GRASP65; Golgi apparatus showed similar morphological alterations with that in H2O2injury group at6h, and the neurons with Golgi morphological alterations showed no significant differeces compared to that in H2O2injury group(P>0.05).7. Flow cytometric analysis results showed no significant early stage cell apoptosis both in H2O2injury group and in U0126inhibited group (apoptotic rate<0.1%), and each group showed no significant difference with the control group (P=NS). However, cell mortality (late stage apoptotic/necrotic cells) increased both in H2O2injury group and in U0126inhibited group, with significant differences compared to that in control group (P<0.01), and U0126obviously reduced cell mortality at each time point compared with that in H2O2injury group (P<0.05). These results showed that the inhibition of ERK activation rescued N2a from cell death in oxidative stress.Conclusion:1. ERK pathway was activated in oxidative stress and was involved in oxidative stress-mediated cell death; inhibition of ERK activation could play a protective role in this process;2. GRASP65was phosphorylated in oxidative stress by ERK;3. Golgi apparatus experienced irregular morphological alterations in H2O2-induced oxidative stress, but which may not be associated with ERK mediated GRASP65phosphorylation or cell death.更多还原