【作者】 黄金莎；

【导师】 王涛；

【作者基本信息】 华中科技大学 ， 神经病学， 2009， 博士

【摘要】 第一部分鱼藤酮对GAPDH的表达、定位及聚集的影响第一节鱼藤酮对GAPDH的表达、亚细胞定位及糖酵解活性的影响目的研究鱼藤酮对PC12细胞内糖酵解关键酶GAPDH的表达、亚细胞定位及糖酵解活性的影响。方法鱼藤酮作用于体外培养的神经生长因子诱导分化的PC12细胞，采用FITC-Annexin V／PI染色，用流式细胞仪检测细胞凋亡水平；通过实时荧光定量PCR(real-time PCR)和蛋白免疫印迹分析(Western blot)检测鱼藤酮对PC12细胞内GAPDH mRNA和蛋白表达水平的影响；使用亚细胞组分分离及Western blot观察GAPDH的亚细胞定位改变；根据比尔定律采用分光光度法测定鱼藤酮处理后细胞内GAPDH糖酵解活性的改变。结果流式细胞仪检测结果显示，鱼藤酮可时间依赖性的诱发PC12细胞凋亡。实时荧光定量PCR及蛋白免疫印迹分析显示，鱼藤酮处理后细胞内GAPDH mRNA和蛋白表达水平均随时间的延长呈升高趋势；而随处理时间的延长，GAPDH的糖酵解酶活性反而下降；亚细胞组分分离及Western blot进一步证实，鱼藤酮处理后GAPDH主要在细胞核及线粒体组分内增多。结论鱼藤酮可以诱发GAPDH的过表达、抑制GAPDH糖酵解活性并导致GAPDH亚细胞定位的改变，这可能是鱼藤酮诱导的多巴胺能神经元凋亡的重要机制之一。第二节鱼藤酮导致GAPDH的聚集及可能的原因目的探讨鱼藤酮对GAPDH聚集的影响及可能的原因。方法鱼藤酮作用于体外培养的神经生长因子诱导分化的PC12细胞，使用激光共聚焦荧光显微镜观察细胞中GAPDH的分布和性状改变；用非还原型SDS-PAGE检测细胞内GAPDH二硫化物多聚体的变化；分别提取胞浆内去垢剂可溶及去垢剂不溶的蛋白，使用Western blot检测细胞内GAPDH可溶状态的改变。结果激光共聚焦结果显示，正常细胞内GAPDH蛋白均匀分布于胞浆，在细胞核内仅有少量分布。鱼藤酮处理后，胞浆内GAPDH染色增强，且出现明显的GAPDH染色阳性的聚集颗粒，部分细胞胞核内也出现GAPDH的团块状聚集。非还原型SDS-PAGE显示鱼藤酮处理可时间依赖性的增加细胞内二硫化键连接的GAPDH。随鱼藤酮处理时间的延长，细胞内的GAPDH去垢剂可溶及去垢剂不溶的GAPDH均增多，提示细胞内GAPDH可能出现了可溶性质的改变并进一步产生聚集。结论鱼藤酮可以导致细胞内GAPDH的颗粒状聚集。其聚集的可能原因是分子间二硫键的相互连接形成多聚体，并进一步聚集形成去垢剂难溶的蛋白。这可能是鱼藤酮诱导的神经元凋亡及包涵体形成的重要机制。第二部分干扰GAPDH的过表达对多巴胺能神经元的影响及作用机制目的研究RNA干扰(RNAi)技术能否有效抑制GAPDH的表达以及对鱼藤酮诱导的细胞凋亡的影响及机制。方法在实验过程中均添加1mM的丙酮酸以代偿干扰引起的GAPDH糖酵解酶活性的改变。使用脂质体lipofectamine2000将体外化学合成的针对GAPDH基因的小干扰RNA(siRNA)转染进入细胞，使用FAM标记的siRNA进行转染效率的检测，通过实时荧光定量PCR(real-time PCR)和蛋白免疫印迹分析(Western blot)进行干扰效果的鉴定；使用激光共聚焦荧光显微镜观察干扰对鱼藤酮诱导的GAPDH定位和性状的影响；罗丹明123染色流式细胞仪检测线粒体膜电位的变化，Annexin V及PI双染检测细胞凋亡比率。结果实时荧光定量PCR及蛋白免疫印迹分析显示，化学合成的siRNA可有效抑制细胞内GAPDH mRNA和蛋白水平的表达；激光共聚焦荧光显微镜结果显示，干扰GAPDH的表达可减少鱼藤酮所致的GAPDH的聚集及核转位；流式细胞仪检测发现干扰GAPDH表达可明显减少细胞线粒体膜电位的下降和细胞凋亡。结论RNA干扰可减少鱼藤酮诱发的GAPDH的过度表达，减少鱼藤酮诱发的GAPDH的颗粒状聚集及线粒体膜电位的下降和细胞凋亡。第三部分鱼藤酮帕金森病大鼠模型的建立及GAPDH的表达和活性改变第一节鱼藤酮慢性皮下注射及立体定向注射帕金森病大鼠模型的建立及评价目的通过鱼藤酮慢性皮下注射及鱼藤酮立体定向注射建立帕金森病大鼠模型，并进行行为学及组织学鉴定。方法采用背部皮下注射鱼藤酮及立体定向注射鱼藤酮的方法建立帕金森病大鼠模型，使用bar test、grid test、阿扑吗啡诱导的旋转行为观察大鼠的行为学改变；使用HE染色、α-synuclein、泛素(ubiquitin，UB)等抗体染色观察鱼藤酮的外周毒性及细胞内包涵体的形成情况，使用酪氨酸羟化酶(tyrosine hydroxylase，TH)抗体检测大鼠中脑黑质多巴胺能神经元的表达变化。结果鱼藤酮背部皮下注射及立体定向注射均可诱导大鼠出现行动迟缓、自主活动减少等行为学改变，bar test、grid test均显示鱼藤酮处理的大鼠运动潜伏期较对照组明显延长。阿扑吗啡也可诱导立体定向鱼藤酮注射的大鼠出现明显的旋转行为。免疫组化结果显示，两种模型均能导致大鼠中脑黑质内酪氨酸羟化酶免疫活性的下降，并出现α-synuclein和泛素染色阳性的嗜酸性包涵体。此外，在鱼藤酮背部皮下注射的大鼠还观察到明显的外周器官毒性，如肝脏炎症细胞浸润、肾脏局部缺血坏死等，而立体定向注射组未观察到明显的外周毒性。结论鱼藤酮背部皮下注射及立体定向注射均可成功的制作帕金森病大鼠模型。第二节鱼藤酮帕金森病大鼠模型中GAPDH的表达及活性改变目的探讨鱼藤酮帕金森病大鼠模型中GAPDH的表达、酶活性及定位的改变。方法采用鱼藤酮背部皮下注射及立体定向注射的方法，建立鱼藤酮帕金森病大鼠模型。使用免疫组织化学方法观察大鼠皮质、海马、黑质、纹状体等部位GAPDH的表达和定位变化；分光光度计测量各组模型中各脑区内GAPDH糖酵解酶活性的改变。结果在鱼藤酮处理的大鼠中，黑质区神经元胞浆内出现GAPDH表达增高，尤以鱼藤酮立体定向注射的大鼠更为明显。在立体定向注射的大鼠中，还出现GAPDH在胞核内的聚集。而慢性鱼藤酮注射不仅抑制大鼠黑质、纹状体、皮质等部位的GAPDH糖酵解活性，对外周脏器如肝脏、肾脏的GAPDH糖酵解活性也有明显抑制作用。鱼藤酮立体定向模型中则仅发现有黑质及纹状体部位GAPDH糖酵解活性的抑制，外周器官GAPDH酶活性不受影响。结论鱼藤酮可以诱导大鼠中脑黑质GAPDH的过表达，并抑制GAPDH糖酵解活性，这可能是鱼藤酮导致的中脑黑质多巴胺能神经元变性的原因之一。第四部分司来吉兰对鱼藤酮诱导的帕金森病模型的保护作用及机制第一节司来吉兰对鱼藤酮处理的PC12细胞的保护作用及机制目的研究司来吉兰对鱼藤酮处理的PC12细胞的保护作用及机制。方法采用四甲基偶氮唑盐比色法(MTT)检测细胞活力，选定药物干预浓度；通过荧光定量PCR(real-time PCR)和蛋白免疫印迹技术(Westem blot)检测司来吉兰处理对鱼藤酮诱导的GAPDH mRNA和蛋白过表达的影响；根据比尔定律采用分光光度法测定细胞内GAPDH糖酵解活性的改变；激光共聚焦荧光显微镜、亚细胞组分分离及Westem blot观察GAPDH的亚细胞定位和性状改变，流式细胞仪检测线粒体膜电位和细胞凋亡。结果MTT结果显示，10nM～250μM浓度范围内的司来吉兰具有细胞保护作用。实时荧光定量PCR及蛋白免疫印迹分析显示，司来吉兰可减少鱼藤酮诱导的GAPDHmRNA和蛋白的过表达，减少GAPDH糖酵解酶活性的下降；激光共聚焦荧光显微镜显示，司来吉兰可减少GAPDH在胞浆内的聚集及细胞核转位；亚细胞组分分离及Western blot进一步证明，司来吉兰可减少GAPDH在细胞核及线粒体组分内的富集。流式细胞仪检测发现司来吉兰可减少细胞线粒体膜电位的下降和细胞凋亡。结论司来吉兰可抑制GAPDH的过表达、减少GAPDH糖酵解活性的下降，减少GAPDH在细胞核和线粒体内的聚集及线粒体膜电位的下降，这可能是司来吉兰发挥神经保护作用的重要机制。第二节司来吉兰对鱼藤酮诱导的帕金森病大鼠模型的保护作用及机制目的探讨司来吉兰对鱼藤酮诱导的帕金森病大鼠模型的保护作用及机制方法动物随机分为葵花油皮下注射组、鱼藤酮皮下注射组、鱼藤酮皮下+司来吉兰注射组；DMSO立体定向组、鱼藤酮立体定向组、鱼藤酮立体定向+司来吉兰注射组。使用bar test、grid test、阿扑吗啡诱导的旋转行为观察各组大鼠的行为学改变；使用HE染色观察外周毒性，使用免疫组织化学方法观察大鼠皮质、海马、黑质、纹状体等部位GAPDH的表达和定位改变，分光光度计测量各组模型中各脑区GAPDH糖酵解酶活性的变化。结果鱼藤酮皮下注射及立体定向注射均可导致大鼠出现行动迟缓，动作潜伏期延长等帕金森病症候群。免疫组化染色显示，鱼藤酮可以导致大鼠中脑黑质酪氨酸羟化酶活性的下降及GAPDH的过表达。而司来吉兰处理可改善大鼠的行动迟缓、动作潜伏期延长等行为学症状，减少大鼠黑质神经元胞浆内GAPDH阳性物质浓染聚集及胞核内GAPDH的聚集，也可部分缓解大鼠黑质、纹状体等部位的GAPDH糖酵解活性的下降。在皮下注射组，司来吉兰还可以减缓鱼藤酮导致的动物外周器官如肝脏、肾脏内GAPDH糖酵解活性的下降。结论司来吉兰可以通过减少GAPDH的过表达，减少GAPDH糖酵解活性的损伤发挥神经保护作用。更多还原

【Abstract】 PART 1 The Effect of Rotenone on Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) Expression, Subcellular Localization andAggregationSection 1 The Effect of Rotenone on GAPDH Expression, Subcellular Localizationand Enzyme ActivityObjective To investigate the effect of rotenone on GAPDH expression, subcellularlocalization and enzyme activity.Methods Nerve growth factor (NGF) differentiated PC12 cells were treated withrotenone for different periods.The cell apoptotic rates were measured using flow cytometryby FITC-Annexin V/PI staining; Real time-PCR and western blot were used to detect theexpression of GAPDH mRNA and protein level; Subcellular fractionation and western blotwas used to study the subcellular location of GAPDH in cells; GAPDH glycolysis activitywas measured by spectrophotometry.Results Flow cytometry analysis indicated that rotenone induced PC12 cell apoptosis in atime-dependent manner.Real-time PCR and western blot showed that GAPDH mRNA andprotein level were both up-regulated in a time-dependent manner; while spectrophotometrydemonstrated that GAPDH glycolysis activity were decreased after rotenone exposure.Subcellular fractionation and western blot data showed that GAPDH protein was increasedin the nuclear and mitochondria fractions.Conclusions Rotenone might induce GAPDH overexpress, suppress GAPDH glycolyticactivity and induce GAPDH enrich in the nuclear and mitochondria fractions, which mayplay an important role in rotenone induced dopaminergic neuron death. Section 2 Rotenone induce GAPDH aggregation and its possible mechanismObjective To explore the possible mechanism involved in rotenone induced GAPDHaggregation in dopaminergic neurons.Methods Nerve growth factor induced PC12 cells were treated with rotenone.LaserScanning Confocal Microscope (LSCM) was used to observe the GAPDH distribution andcomformational change after rotenone treatment.Non-reduced SDS-PAGE Was used todetect the disulfide-bonded GAPDH in cells.The Triton X-100 soluble and insolubleGAPDH were separated and detected by western blot analysis.Results GAPDH distributed uniformly in the cytosol and seldom GAPDH were locatedin the nuclear in control cells.After rotenone treatment, GAPDH expression was muchhigher, some GAPDH positive aggregates were observed in the cytosol.Non-reducedSDS-PAGE also indicated that rotenone induced a time-dependent disulfide-bondedGAPDH overexpression.After rotenone exposing, both Triton X-100 soluble and insolubleGAPDH were increased.Inclusion Rotenone can induce GAPDH aggregation.The possible mechanism may dueto the increase of disulfide-bonded GAPDH and Triton X-100 insoluble GAPDH.This maybe an important mechanism involved in rotenone induced cell apoptosis and inclusionbodies formation.PART 2 The effect of targeting inhibition of GAPDH expression byRNA interference on rotenone induced cell deathObjective To investigate the effect of GAPDH siRNA on rotenone induced cell apoptosis.Methods The small interfering RNA (siRNA) specific targeted to GAPDH gene was constructed by chemical synthesis.SiRNA was transfected into nerve growth factordifferentiated PC12 cells using lipofectamine2000.Transfection efficiency was detectedusing FAM-labeled siRNA.Interfere efficiency was identified by real-time PCR andwestern blot analysis.Laser scanning confocal microscope was used to study the locationand aggregation of GAPDH.The mitochondria membrane potential changes of transfectedor unvalidated negative siRNA transfected cells after rotenone exposure were detected byrhodamine 123 staining using flow cytometry.Results Real-time PCR and western blot analysis showed that GAPDH siRNA canefficiently suppress GAPDH expression.LSCM found that GAPDH became aggregated inthe cytosol and nuclear translocation in rotenone treated PC12 cells; while GAPDH siRNAtreated cells has lesser GAPDH aggregation and nuclear translocation.As detected by flowcytometry, down-regulation of GAPDH significantly decreased rotenone inducedmitochondrial membrane potential collapse, compared with unvalidated negative control.Conclusions Down-regulation of GAPDH suppresses rotenone induced GAPDHaggregation and nuclear translocation, decreases rotenone induced mitochondrialmembrane potential collapse and cells apoptosis, which may play a role in rotenoneinduced dopaminergic neuron death.PART 3 GAPDH Expression and Activity in Rotenone-induced PD RatsSection 1 Rotenone-induced PD models: a comparison between subcutaneousinjection and stereotactic injectionObjective To explore and compare the rotenone-induced PD models: subcutaneousinjected models and stereotactic injected models.Method Rotenone was injected subcutaneously at the back or stereotactically at the VTA and SN of the rat brain.The behaviors features of animals were investigated using bar test,grid test and apomorphine-induced rotations.HE pathology, the immunoreactivity oftyrosine hydroxylase,α-synuclein, and ubiquitin in the brains were observed byimmunohistochemistry.Results Behavior investigation indicated that both rotenone subcutaneous injection andstereotactic injection can induce Parkinsonism features.Immunohistochemistry studysuggested that the tyrosine hydroxylase immunoreactivity were decreased in subtantia nigra.Acidophily inclusion bodies andα-synuclein-, ubiquitin-positive inclusions were found inrotenone-induced PD models.Organ toxicity was observed in subcutaneously injected PDmodels but not in stereotactically injected PD models.Conclusion Both rotenone subcutaneous injection and stereotactic injection are effectiveto induced PD models.Section 2 GAPDH expression and activity in rotenone-induced PD modelsObjective To observe the expression, distribution and enzyme activity of GAPDH inrotenone-induced PD rats.Methods Rotenone was injected subcutaneously at the back or stereotactically at theVTA and SN of the rat brain to induce PD models.Grid test, bar test andapomorphine-induced rotations were used to detect the behaviour changes inrotenone-induced PD models.Immunohistochemistry study was used to investigate theexpression and distribution of GAPDH in rotenone-induced PD models.GAPDH glycolyticactivity in substatia nigra, striatum and other brain areas were studied byspectrophotometry.Results In rotenone-induced PD models, GAPDH was up-regulated in the substatia nigraespecially in rotenone stereotactically injected PD models.Some neurons existed GAPDHnuclear translocation and aggregation.The GAPDH glycolytic activity was not only significantly decreased in the substatia nigra but also the peripheral organs such as the liverand kidney in subcutaneously rotenone administered rats.While there was only GAPDHactivity decrease in substatia nigra and striatum but not in peripheral organs in rotenonestereotacitcally injected models.Inclusions GAPDH protein is up-regulated and aggregated in the brain of rotenonetreated rats, but the glycolytic activity of GAPDH is decreased.PART 4 Effect of Deprenyl on Rotenone-Induced PD Models and ItsPossible MechanismSection 1 The protective effect of deprenyl on rotenone treated PC12 cells and thepossible mechanismObjective To investigate the protective effect of deprenyl on rotenone induced cell deathand its possible mechanism.Methods NGF differentiated PC12 cells were treated with deprenyl at differentconcentrations (1nM-1mM) instantly after rotenone treatment.Cell viability was measuredby MTT.The expression of GAPDH mRNA and protein were detected using real time-PCRand western blot.GAPDH glycolysis activity was measured by spectrophotometry.LaserScanning Confocal Microscope (LSCM) and subcellular fractionation and western blot wasused to study the subcellular location of GAPDH in deprenyi treated and untreated cells.The cell apoptosis were detected using flow cytometry.Results Cell viability declined in a concentration-dependent manner after rotenoneexposure, while 10nM-250μM deprenyl can partly inhibited rotenone induced cell death.The GAPDH mRNA and protein level were both significantlY decreased after deprenyltreatment compared with rotenone treated groups.Spectrophotometry analysis demonstrated that GAPDH glycolysis activity was increased after deprenyl exposure.Subcellular fractionation and western blot further indicated that deprenyl can reducerotenone induced GAPDH nuclear and mitochondria translocation.Flow cytometry alsoshowed that deprenyl exposure can reduce rotenone induced mitochondrial membranepotential collapse and cells apoptosis.Conclusions Deprenyl can reduce rotenone induced GAPDH overexpression, nuclear andmitochondria translocation, increase GAPDH glycolytic activity and protect cells fromapoptosis.Section 2 The effect of deprenyl on rotenone-induced PD ratsObjective To investigate the effect of deprenyl on rotenone-induced PD rats and itspossible mechanism.Methods Rotenone was injected subcutaneously at the back or stereotactically at theVTA and SN of the rat brain to induce PD models.Deprenyl were injected intraperitoneallyafter rotenone exposure.Grid test, bar test and apomorphine-induced rotations were used todetect the behaviour changes in animals.Immunohistochemistry study was used to studythe GAPDH expression and distribution in each group.GAPDH glycolytic activity insubstatia nigra, striatum and other brain areas were studied by spectrophotometry.Results In rotenone-induced PD models, GAPDH was up-regulated in the substatia nigraespecially in rotenone stereotactically injected PD models.Deprenyl can reduce rotenoneinduced GAPDH overexpression in rat brains.The GAPDH glycolytic activity wasdecreased in the substatia nigra, striatum and the peripheral organs in subcutaneouslyrotenone administered rats, while there was only GAPDH activity decreases in substatianigra and striatum but not in peripheral organs in rotenone stereotacitcally injected models.Inclusions Deprenyl can relieve GAPDH overexpression and the decrease of GAPDH glycolytic activity induced by rotenone exposure, and have protective effect onrotenone-induced PD rats.更多还原