【作者】 李婷；

【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：氧化应激在脑缺血后神经元的损伤中起关键作用,是造成脑缺血再灌注损伤的主要因素。随着疾病的研究深入到亚细胞水平,越来越多的研究证实与亚细胞器相关的细胞死亡是一个值得重视的领域。内质网、线粒体及溶酶体等在氧化应激相关细胞死亡中的主导作用已得到广泛认可及深入研究。近年来,针对高尔基体在氧化应激中的研究发现,同线粒体及内质网相似,高尔基体也能够感知并传导氧化应激信号即高尔基体应激(Golgi Apparatus Stress,GAS)。通过发现并阻断不恰当的GAS将成为防止或治疗氧化应激相关神经系统疾病的研究热点。虽然GAS与疾病的研究已经得到了越来越多的重视,针对高尔基体在氧化应激中的作用也已经进行了一系列卓有成效的研究,但目前仍处于初期阶段,缺乏可以检测并评估的相关因子。如内质网应激过程中,可以检测到伴侣蛋白GRP78表达的上调、蛋白水解酶Caspase-12及促凋亡编码基因CHOP等的激活以及线粒体应激中,活性氧基ROS的生成、线粒体膜电位的改变、细胞色素C的释放以及Bcl-2家族因子的变化等等。我们推测在氧化应激所诱导的GAS过程中,也存在与其功能及结构调适相关联的因子或基因,能够提示GAS的存在,动态反映应激水平,并受其他细胞器信号通路的影响,或者在高尔基体自我适应失败后能直接触发特异性凋亡通路。该因子应该具备以下特点：①来源于高尔基体,具有与高尔基体功能密切相关的活性；②在氧化应激作用下能迅速反应性上调,并随应激水平的改变而发生变化；③对高尔基体的功能或下游凋亡信号有直接影响。GOLPH3是一类新的高尔基体外膜蛋白,具备成为GAS相关因子的潜质。因此,本研究的目的旨在利用体外细胞氧糖剥夺复氧(OGD/R)模型探索在脑缺血再灌损伤中,GOLPH3与氧化应激的关系,对高尔基体结构及分泌功能的影响,是否对高尔基体、内质网、线粒体应激及凋亡、自噬通路产生影响；进一步完善高尔基体应激学说的理论基础。研究方法：1.培养小鼠神经细胞瘤N2A细胞,建立氧糖剥夺再灌注(OGD/R)模型,并采用光学显微镜、酶学检查等评价OGD/R模型；2.实验分为正常对照组、普通OGD/R组、保护性药物干预后OGD/R组,细胞毒性药物干预组；其中普通OGD/R组、药物干预后OGD/R组按时间点不同分为41H、12H、24H、48H四个亚组；保护性干预药物为抗氧化剂N-乙酰半胱氨酸(N-acetylcysteine, NAC)以及内质网应激特异性阻断剂4-苯基丁酸钠(4-phenylbutyrate acid,4-PBA);细胞毒性药物为能造成钙超载的毒胡萝卜内酯(Thapsigargin,TG)及N-甲基-D-天冬氨酸(NMDA);3. Western blot检测各组中GOLPH3, GRP78, LC3Ⅰ、Ⅱ亚基,p75NTR的蛋白表达变化；4.RNA干扰技术抑制GOLPH3表达,ShRNA的筛查及载体转染;5.流式细胞仪测定各组中细胞凋亡率的变化(Annexin V-FITC/PI);6.荧光法(DCFH-DA)测定各组中ROS生成的变化；7. ELISA法检测细胞上清BDNF浓度；8.免疫荧光共聚焦方法观察GOLPH3的细胞亚定位及变化。结果：1. OGD/R后细胞形态的变化、ROS生成及GRP78表达的检测显示：随着氧糖剥夺后复氧时间的延长,小鼠N2A细胞形态结构发生显著变化,与正常组比较ROS生成显著增加(P<0.01),GRP78表达明显增加(P<0.01),表明复氧后的再灌注氧化应激损伤诱导线粒体及内质网应激的发生,加剧了细胞损伤；2.抗氧化药物干预后再予以氧糖剥夺／复氧4小时、24小时后观察细胞形态变化、ROS生成、GRP78表达及凋亡率的变化,结果提示：N2A细胞形态变化较干预前不明显,4-PBA或NAC均能显著减少OGD/R24H组N2A细胞ROS的生成(P<0.01), GRP78表达在4-PBA干预后的OGD/R4H (P<0.01)、24H组(P<0.05)均有下降,仅在NAC干预后的OGD/R4H组稍有下降(P<0.05);4-PBA或NAC均能显著减少OGD/R24H组的细胞凋亡率(P<0.01)。表明抗氧化预处理或改善内质网未折叠蛋白反应(UPR)均能减轻缺血再灌注诱发的氧化应激损伤；3.细胞毒性药物干预对细胞形态及氧化应激水平的影响：作为阳性对照,实验中还应用到可以造成钙超载的细胞毒性药物TG或NMDA对N2A细胞进行干预。在二者分别干预N2A细胞3小时后,我们发现细胞形态发生了明显变化伴随ROS生成及GRP78表达的显著升高,早期及晚期凋亡率也明显增加(P<0.01),各项数值均超过OGD/R模型组的最高值。说明药物性钙超载诱导的细胞氧化应激程度更为强烈,对细胞的损伤更直接；4.GOLPH3表达与氧化应激：在氧糖剥夺／复氧(OGD/R)后各时间点,N2A细胞GOLPH3的表达较正常组显著上升(P<0.01),且上升程度与OGD/R复氧时间相关。而抗氧化药物4-PBA或NAC干预后的OGD/R4H组中GOLPH3也出现表达的下降(P<0.05)。细胞毒性药物TG或NMDA已证实能引起强烈的氧化应激损伤,二者干预后GOLPH3的表达均较正常对照组明显增强(P<0.01)。结果提示氧化应激显著诱导GOLPH3的表达,其表达水平与氧化应激程度呈现正相关；5.GOLPH3细胞内定位的变化：共聚焦显微镜下观察到OGD/R4小时再灌注损伤后标记GOLPH3的绿色荧光由正常状态下紧凑的囊泡样结构逐渐向胞浆弥散开,胞浆的染色增多,随着复氧时间的延长,到OGD/R复氧24小时后高尔基体囊泡样结构更弥散,部分呈点状散在于整个细胞质中,伴随荧光强度的增加。在OGD/R模型中,随着应激程度的加重,GOLPH3除了表达的明显上调外,还有胞内定位的改变,与高尔基体碎裂发生相同的形态变化。细胞毒性药物TG或NMDA已证实能引起神经元高尔基体的碎裂,二者干预后GOLPH3的荧光强度均明显增强(P<0.01),由核周的紧凑囊泡样结构变为胞浆中的点状弥散,少量片状浓染,与这两种细胞毒性药物所致的高尔基体碎裂现象相吻合；6. GOLPH3ShRNA载体转染：GOLPH3-mus-505的转染能够显著抑制GOLPH3RNA水平,而在蛋白质水平抑制效率不如RNA水平高。正常情况下,GOLPH3的表达较对照组下降了33.6%(P<0.05),而经历OGD/R复氧4小时后,GOLPH3表达的增加不如对照组明显,相比下降了约46.6%(P<0.01),OGD/R复氧24小时后,ShRNA转染对GOLPH3蛋白水平的抑制为37.8%(P<0.01)；7. ShRNA干扰研究证实GOLPH3能促进OGD/R复氧24小时组神经生长因子低亲和力受体p75NTR的降解(P<0.05),显著减少24小时组N2A细胞脑源性神经生长因子BDNF的分泌(P<0.01)；8. ShRNA干扰研究证实GOLPH3能影响OGD/R模型早期自噬体形成：GOLPH3ShRNA干扰后,OGD/R复氧4H组LC3II/I比值较正常对照组及同时间点对照组均有显著上升(P<0.01),而OGD/R复氧24H组则显著低于同时间对照组(P<0.01),说明GOLPH3对自噬的影响与应激水平相关；9. ShRNA干扰研究证实GOLPH3能促进OGD/R复氧24小时组ROS的产生(P<0.01)以及N2A细胞的凋亡率(P<0.01),但并不影响GRP78的表达,提示GOLPH3在氧化应激过程中促进线粒体ROS生成,并最终促进凋亡率；结论：1.GOLPH3是高尔基体来源的应激相关蛋白,与细胞内氧化应激水平呈现正相关；2. OGD/R模型中GOLPH3通过减少BDNF的分泌以及促进p75NTR的降解介导高尔基体应激相关信号参与到氧化应激中,并通过促进ROS的生成以及影响应激后早期自噬体的形成,最终促进凋亡的发生。更多还原

【Abstract】 Objective:It is widely accepted that the oxidative stress is the major cause of neuronal death in brain ischemia. Increasing evidence implicate organelle-dependent initiation of Oxidative stress related cell death is a research area worthy of pursuing. Recently, researchers suggest that the Golgi apparatus like ER and Mitochondria also presents an exciting role in stress sensing of cell death pathways and acts as a common downstream-effecter, which undergoes disassembly and fragmentation during apoptosis in several neurological disorders, and described it by the term of "GA stress" for the first time.Mammalian GOLPH3, also known as GPP34/GMx33/MIDAS, is an exciting new class of Golgi outer membrane protein involved in vesicular trafficking, Golgi architecture maintaining, receptor sorting, protein glycosylation and cell signaling pathways that localize to the cytoplasmic face of the trans-Golgi. We predict the possibility that GOLPH3for its diverse functions closely related with Golgi may exist as a stress-related Golgi-related protein that is mobilized during GA stress response.We have successfully used a cell culture model of oxygen and glucose deprivation following reoxygen (OGD/R) to mimic the ischemia/reperfusion injury in vitro. Our studies will focus on the role of GOLPH3in Golgi stress-sensing and the functional significance of GOLPH3in oxidative stress response of GA during ischemia/reperfusion injury caused by OGD/R model.Experimental Methods:1. We employed a stable OGD/R model to mimic ischemic-like conditions in vitro. The neuronal injury was measured by the morphologic changes studied under Light Microscopes and detection of cell apoptosis;2. Experimental grouping was designed as follows:control group; toxic drug intervention group (Thapsigargin/TG or NMDA); common OGD/R group; OGD/R group with drug intervention (N-acetylcysteine/NAC or4-phenylbutyrate acid/4-PBA). The latter two groups were divided into four different subsets for various reoxygen time points following4hours’OGD (OGD/R4H,12H,24H and48H);3. The protein level of GOLPH3, GRP78, LC3Ⅰ/Ⅱ, p75NTR was determined by western blotting;4. GOLPH3ShRNA were designed and transfected into N2A cells by Lipofectamine2000, GOLPH3RNA level were analyzed by RT-PCR;5. The rates of apoptosis were measured by flow cytometric analysis; 6. The intracellular ROS production was detected by fluorescence method;7. The concentration of BDNF in cell supernatant was analyzed by ELISAkit;8. Confocal Immuno-fluorographs of GOLPH3and DAPI were taken to determine the sub-cellular location of GOLPH3.Result:1. The ROS detection and GRP78expression as well as the morphology change of N2A cells showed that the stress reaction of ER and Mitochondria were strongly induced by OGD/R model and direct injury were brought by severe ischemia/reperfusion;2. The intervention of anti-oxidant drugs (NAC or4-PBA) before OGD/R resulted in significant decrease in ROS production of24H group (P<0.01). While, GRP78expression in4H (P<0.01) or24H (P<0.05) groups was decreased after4-PBA intervention, down-regulation of GRP78was only detected in4H group after NAC intervention;3. The intervention of cytotoxic drugs by Thapsigargin/TG or NMDA significantly increased the ROS production and GRP78expression in N2A cells (P<0.01);4. GOLPH3expression were induced by OGD/R model in a time-dependent manner, increased remarkably in OGD/R groups than in control (P<0.01). The intervention of NAC or4-PBA both decreased the expression of GOLPH3in OGD/R4H group (P<0.05). Correlation analysis indicated that GOLPH3was highly co-related with ROS and GRP78in OGD/R model; Cytotoxic drugs intervention of TG or NMDA induced the highest expression level of GOLPH3(P<0.01)5. Confocal Immuno-fluorographs showed that the GOLPH3labeled Golgi apparatus localized to a compact peri-nuclear ribbon, consisting of multiple vesicle-like structures arranged in close opposition to each other to form stacks. Meanwhile, a little amount of G3staining diffused in the cytoplasm. In contrast, after24-hour reoxygenation following24-hour OGD exposure, the compact vesicle-like structures by G3staining fragmented into punctuate-like structures, dispersed throughout the cytoplasm with increased intensity; TG or NMDA intervention which can induce Golgi Fragmentation here also induced notable sub-cellular apposition change of GOLPH3into intensive fragments;6. RNAi-Mediated Silencing research of GOLPH3by mus-505ShRNA could effectively inhibit the RNA as well as its protein level of GOLPH3in N2A cells;7. RNAi-Mediated Silencing research found out that GOLPH3promote the degradation of p75NTR and decrease the secretion of BDNF by N2A cells in OGD/R24H group (P<0.01);8. A time-dependent elevation of autophagy level after OGD/R injury were detected by LC3II/I ratio, GOLPH3suppression could induce the LC3II/I ratio of OGD/R4H group (P<0.05), but inhibit that of24H group (P<0.01).9. RNAi-Mediated Silencing of GOLPH3research demonstrated that in OGD/R24H group GOLPH3promote ROS production and increase the apoptosis rates of N2A cells, mediate a specific apoptotic pathway of Golgi apparatus Stress (GAS) during oxidative stress (P<0.01);Conclusion:1. GOLPH3is a Golgi-originated stress-related protein; Its expression is closely related with the level of oxidative stress;2. By inhibiting the secretion of BDNF, promoting the degradation of p75NTR, and affecting the formation of stress-related primary autophagosome, GOLPH3mediates Golgi apparatus Stress (GAS) to participate in OGD/R model induced oxidative stress.更多还原