【作者】 新吉乐；

【导师】 周秋丽； 张扬；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 背景和目的帕金森病（Parkinson’s disease，PD）是一种中老年常见的神经系统退行性疾病，其发病率的上升不仅严重影响患者的生活质量，而且给家庭和社会带来沉重的负担。PD的临床症状包括静态下震颤、反应迟钝、僵硬和姿势扭曲等。PD的主要病理特点是黑质致密部的多巴胺能神经元丢失，进而使纹状体内的多巴胺水平降低。目前，PD的发病原因尚不完全清楚。各类体内和体外实4验显示，PD的发病机制涉及线粒体功能障碍、氧化应激、泛素化蛋白酶体等途径。最近的研究表明，PD发病机制还涉及未折叠蛋白聚积引起的内质网应激，即内质网应激（endoplasmic reticulum stress, ER stress）介导的细胞凋亡参与PD的发病过程。因此，深入了解PD发病机制，探寻有效的神经保护剂是目前治疗PD的重要策略。PD发病机制的探讨和潜在治疗药物的筛选及评价，在很大程度上依赖于疾病相关体内外模型的建立及应用，特别是离体细胞模型的建立及应用。众所周知，PD是一种慢性疾病，具有缓慢、进展性的发病过程和特征，但是目前国内用于PD发病机制和药物筛选的离体细胞模型大多为急性损伤模型。我们的实践证明，采用常规急性损伤细胞模型已经不能客观反映PD缓慢、渐进性发展的规律和特征，必须建立PD稳定的慢性损伤的体外模型，以适应PD发病机制研究及探寻有效神经保护剂的需要。基于以上的理解，本研究首先以1-甲基-4-苯基吡啶离子（1-methyl-4-phenylpyridinium, MPP~+）作为诱导剂，以SH-SY5Y细胞作为模型载体，建立PD急、慢性细胞损伤模型，确定建立急、慢性细胞模型的最佳条件。在比较急、慢性SH-SY5Y细胞损伤的规律和特点基础上，利用慢性SH-SY5Y细胞损伤模型探讨内质网应激介导的SH-SY5Y细胞凋亡机制。并在上述研究基础上观察鹿茸多肽（Velvet antler polypeptides, VAPs）对SH-SY5Y细胞凋亡的干预作用及初步机制探讨，为进一步开发抗PD的药物提供科学依据和研究思路。方法1.以新鲜梅花鹿茸为原材料，通过胶体磨匀浆、硫酸铵沉淀、冷冻干燥等方法制备VAPs；进一步利用SDS-聚丙烯酰胺凝胶电泳（polyacrylamide gelelectrophoresis，PAGE）、激光解吸电离飞行时间质谱和氨基酸组成分析等技术初步分析鹿茸总肽的理化性质。2.采用MPP~+诱导人神经母细胞瘤SH-SY5Y细胞发生凋亡，设置不同浓度MPP~+分别作用24,48,72h，建立MPP~+急性损伤细胞模型；MTT法检测细胞活力，最终确定120.9μM MPP~+作用72h为建模浓度和时间。进而将培养的SH-SY5Y细胞分为空白对照组、MPP~+组和VAPs组。对照组给予细胞培养基培养，MPP~+组给予120.9μM的MPP~+处理，VAPs组给予不同浓度VAPs（62.5、125、250μg/mL）与120.9μM MPP~+同时处理。采用Hoechst33342染色检测细胞凋亡，罗丹明123染色检测线粒体膜电位（△Ψm），H2DCFDA染色检测细胞内活性氧簇（reactive oxygen species，ROS）的水平，Western blot和免疫细胞化学检测内质网分子伴侣重链结合蛋白/糖调节蛋白（Heavy-chain bindingprotein/glucose regulated protein78, Bip/GRP78）、CCAAT/增强子结合蛋白同源蛋白（C/EBP homologous protein, CCAAT/enhancer binding protein homologousprotein, CHOP）、磷酸化c-Jun氨基末端激酶/应激活化蛋白激酶（c-Jun N-terminalkinase/Stress-activated protein kinases, JNK/SAPK）、半胱氨酸天冬氨酸蛋白酶12（caspase-12）等4种蛋白的表达情况。3.采用MPP~+诱导人神经母细胞瘤SH-SY5Y细胞发生凋亡，设置不同浓度MPP~+分别作用5,10,15天，建立MPP~+慢性损伤细胞模型；MTT法检测细胞活力。将培养的SH-SY5Y细胞分为空白对照组、MPP~+组和VAPs组。对照组给予细胞培养基培养，MPP~+组给予31.1μM的MPP~+处理，VAPs组给予不同浓度VAPs与31.1μM MPP~+同时处理。上述各组细胞均孵育5,10,15天后，采用MTT法检测细胞存活率。采用Hoechst33342染色检测细胞凋亡，罗丹明123染色检测△Ψm，H2DCFDA染色检测细胞内ROS的水平，Western blot检测GRP78、CHOP、磷酸化JNK（p-JNK）和caspase-12等蛋白表达情况。结果1.鹿茸总多肽（VAPs）所得VAPs为白色粉末状，水溶性较好，蛋白质含量为54%。VAPs分子量主要分布在10kDa和20kDa区域范围内。进一步将分子量10kDa区域的多肽进行质谱分析，其分子量在3000～11000Da范围内。VAPs主要由天冬氨酸、丝氨酸、谷氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、苯丙氨酸、赖氨酸、精氨酸和脯氨酸组成，没有检测到半胱氨酸、酪氨酸和色氨酸。2.急性损伤模型⑴120.9μM的MPP~+可以诱导SH-SY5Y细胞存活率下降，凋亡率增加。125、250μg/mL的VAPs可减少细胞凋亡，以72h到达最大保护效应。MPP~+导致△Ψm降低，ROS（活性氧簇）含量增加。125、250μg/mL的VAPs拮抗MPP~+诱导产生的△Ψm和ROS的变化。⑵MPP~+诱导caspase-12表达显著增加，不同浓度VAPs可使MPP~+诱导的Caspase-12表达有所降低。在MPP~+损伤的SH-SY5Y细胞未观察到GRP78、CHOP、p-JNK等内质网应激（ER）相关蛋白的显著表达。综上，MPP~+致SH-SY5Y细胞损伤的主要特征为线粒体功能障碍和氧化应激介导的细胞凋亡，VAPs对该损伤有拮抗和纠正作用。在MPP~+诱导的急性损伤模型中，MPP~+可使SH-SY5Y细胞内的caspase-12水平显著提高，但没有看到其它内质网应激相关蛋白的过表达。内质网应激相关蛋白沉默表达提示，在急性损伤模型中caspase-12的激活及诱导细胞凋亡并非借助于内质网应激相关途径的启动，可能主要介导线粒体凋亡途径及氧化应激凋亡途径。3.慢性损伤模型（1）建立了MPP~+作用SH-SY5Y细胞5天（IC50为31.1μM）、10天（IC50为22.1μM）和15天（IC50为10.4μM）的慢性损伤模型，500μg/mL VAPs能有效提高SH-SY5Y细胞5天、10天和15天的存活率。（2）在MPP~+慢性损伤SH-SY5Y细胞中，没有看到MPP~+急性损伤模型呈现的凋亡特征性改变和△Ψm的变化，VAPs也没有明显作用。但在MPP~+损伤5天的SH-SY5Y细胞内，ROS水平显著升高，VAPs能明显降低细胞内ROS含量。（3）免疫细胞化学和Western blot检测均显示，MPP~+作用5天的SH-SY5Y细胞中，内质网应激相关蛋白caspase-12、GRP78、CHOP和p-JNK表达水平显著提高，VAPs与MPP~+共孵育可明显对抗caspase-12、GRP78、CHOP、p-JNK表达的上调。在MPP~+诱导10、15天的SH-SY5Y细胞中则看不到上述内质网应激相关蛋白的变化，VAPs也没有相应的作用。结果提示，MPP~+致SH-SY5Y细胞慢性损伤的主要特征为内质网应激相关蛋白的激活，是一种功能性损伤。综上，在低浓度MPP~+（31.1μM）诱导5天的慢性损伤细胞模型中呈现出与高浓度MPP~+（120.9μM）诱导72小时急性损伤模型完全不同的损伤模式和特征性改变。研究表明，急性损伤模型是以线粒体功能障碍和氧化应激损伤介导的细胞结构性破坏，镜下看到大量凋亡细胞和细胞坏死残片。而慢性损伤模型则是以内质网应激和氧化应激介导的细胞功能性损伤，镜下没有典型的细胞凋亡和坏死。后者更接近于PD缓慢、进展性疾病的特点，是研究PD内质网应激相关机制的理想模型。鹿茸多肽对MPP~+诱发的SH-SY5Y细胞慢性损伤有保护作用，其机制可能是通过抑制内质网应激相关凋亡分子表达而实现的。更多还原

【Abstract】 BackgroundParkinson’s disease (PD) is a common neurodegenerative disease in agedpopulation. The growing incidence of PD is a socio-economic problem affecting lifequality of PD patients. The syptoms of PD inclue tremor, bradykinesia, rigidity andflexed posture. Pathological features of PD implicated loss of dopaminergic neuronsin substanitial nigra, which cause the decreased dopamine level in the striata. Untilnow, the mechanism underlying PD onset is not fully understood. Most of the in vivoand ex vivo studies had illustrated that some of the mechanism involved in the PDpathogenesis, including mitochondial dysfunction, oxidative stress, ubiquitin-proteasomal pathway, etc. Recently, a misfolded protein related endoplasmicreticulum stress (ER stress) had been most intensively studied for its actions toneuronal cell death. Thus, understanding the process of ER stress is of great helpful toexplore novel neuroprotective agent for PD treatment. Clarifing the mechanisms ofPD onset and development of neuroprotective therapeutics are largely depend onestablishment of disease model, especially model in vitro. It is known that PD is achronic and progressive disease, but acute lesioned model in vitro was most commonused in domestic studies for PD. Our study indicated that the acute lesioned modelfailed to relect the chronic and progessive process of PD onset. Hence, a chroniclesioned model in vitro should be attentioned considerably in order to reveal the actualmechanism of PD and develop novel neuroprotective agents. Based on theseunderstandings,1-methyl-4-phenylpyridinium (MPP~+), a neurotoxin most commonlyused in establishment of PD model in vitro and human neuroblastoma SH-SY5Y cellline had been utilized to establish PD model. In this study, SH-SY5Y cell underoptimized acute or chronic MPP~+exposure was selected in comparison to features ofacute and chronic lesioned model. Based on these data, the chronic MPP~+lesionedmodel was selected to study ER stress mediated cell death, and the neuroprotectiveeffect of velvet antler polypeptides (VAPs), the peptides extracted from cervus nippon,was evaluated to develop the potential therapeutic agent for PD treatment. Methods1. VAPs was obtained from fresh velvet antler using colloid mill, ammonium sulfateprecipitation, lyophilizing procedure. For further study, SDS-polyacrylamide gelelectrophoresis (SDS-PAGE), laser desorption ionization time-of-flight massspectrometry (MALDI-TOF-MS) and amino acid composition analysis wereperfomed to characterize the physical and chemical features of VAPs.2. For acute MPP~+lesioned model in vitro, MPP~+with different concentrations wereexposed to human neuroblastoma SH-SY5Y cell line for24h,48h and72hrespectively. The half maximal inhibitory concentration (IC50) of MPP~+in SH-SY5Yand protective effect of VAPs against MPP~+cytotoxicity were measured using MTTassay. For acute MPP~+exposured model, a72h exposed model was selected. Forfurther study in this model, cells were divided into control, model and VAPs treatedgroup. Control cells were incubated with midium containing10%fetal bovine serum,MPP~+-treated cells were incubated with120.9μM MPP~+, VAPs group cells wereincubated with62.5、125、250μg/mL VAPs in the presence of120.9μM MPP~+. Cellviability was detected with MTT assay; Cell apoptosis rate and mitochondrialmembrane potential (ΔΨm) were measured using Hoechst33342and rhodamine123staining respectively; Intracellular reactive oxygen species (ROS) level was assayedwith H2DCFDA staining; Western blot and immunocytochemistry were performed todetect the level of ER stress related proteins, such as caspase-12, heavy-chain bindingprotein/glucose regulated protein78(Bip/GRP78), C/EBP homologous protein,CCAAT/enhancer binding protein homologous protein (CHOP) and c-Jun N-terminalkinase/Stress-activated protein kinases (JNK/SAPK).3. For establishment of chronic MPP~+intoxication model, SH-SY5Y cells wereexposed to MPP~+for5days,10days and15days. Cell viability was measured withMTT assay; Cell apoptosis rate and ΔΨm were detected with Hoechst33342andrhodamine123staining; Intracellular ROS production was determined withH2DCFDA staining; Western blot and immunocytochemistry were processed forcaspase-12, GRP78, CHOP, phosphorylated JNK（p-JNK). Results1. Velvet antler polypeptides (VAPs)A water soluble fluffy white powder containing54%protein had been obtained. Thelaser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS)analysis indicated that molecular weigh of most of the peptides in VAPs ranged from10kDa~20kDa. Further purification and MALDI-TOF-MS analysis characterizedpeptides with molecular weigh of3~11kDa, which enrihed in asparagine, serine,glutamic acid, glycine, alanine, valine, leucine, phenylalanine, lysine, arginine,proline, but no cystine, tyrosine, tryptophan detected.2. PD model received acute MPP~+exposure⑴120.9μM MPP~+decreased the cell viability of SH-SY5Y cell and increased cellapoptotic rate.125and250μg/mL VAPs protected against MPP~+-induced cell deathin a significant way, which was much apparent for72h protection. MPP~+lead to△Ψ mdecrease and ROS production, but VAPs inhibited these changes in SH-SY5Ycell induced by MPP~+.⑵MPP~+exposure result in a robust expression of caspase-12, and VAPs treatmentinhibited this process. GRP78, CHOP, p-JNK expression were not observed in cellsexposed to MPP~+. Collectively, these results show that a acute MPP~+exposurecharacterized with mitochondrial dysfunction and oxidative sress mediated cell death.VAPs counteract cell death induced by MPP~+. MPP~+incuced caspase-12expression ina significant way, but other RE stress associated proteins such as GRP78, CHOP andp-JNK were not apparent. It is conceivable that an acute MPP~+lesioned model in vitrocaused cell death depending on activation of mitochondrial pathway related caspasecascade and oxidative stress, but not determined by ER stress associated cell deathsignaling.3. Chronic MPP~+intoxication model⑴IC50values for5days,10days and15days MPP~+exposure were31.1μM,22.1μM and10.4μM respectively, which were used to establish MPP~+intoxication modelfor chronic exposure. MTT assay indicated500μg/mL VAPs protected against MPP~+induced decrease of cell viability.⑵In MPP~+intoxication model for chronic exposure, either5days exposure or10days,15days exposure influenced cell apoptosis rate or△Ψm. But ROS productionwas visualized using fluorescent probe in cells exposed to31.1μM MPP~+for5days. 22.1μM MPP~+or10.4μM MPP~+for10days and15days exposure had no effect onROS production. VAPs treatment inhibited the ROS level in MPP~+lesioned model for5days.⑶Western blot and immunocytochemistry indicated there was caspase-12, GRP78,CHOP and p-JNK expression in cells treated with MPP~+for5days. VAPs treatmentdownregulated these protein expression. But neither22.1μM MPP~+,10.4μM MPP~+exposure nor VAPs treatment for10days and15days induced ER stress associatedprotein expression, which indicated the chronic MPP~+exposure lead to ER stressactivation, but not initiation of mitochondrial pathway directly. These data show thatSH-SY5Y cell under31.1μM MPP~+exposure for5days and120.9μM exposure for72h appeared complate different cell signaling phenotypes. Our data identified acuteMPP~+injury induced mitochondrial dysfunction and oxidative stress mediated celldeath, which had been substantiated by cell apoptosis bodies in response to MPP~+exposure. A chronic MPP~+exposure induced ER stress and oxidative stress, but failedto result in cell death. We recognized that a chronic MPP~+intoxication model canmimic the progressive and long term neurodegenerative process of PD onset muchbetter than a acute and short term model. In conclusion, our model in vitro is ideal forstudying ER stress associated pathways. VAPs protect against chronic MPP~+intoxication and the mechanism underlying supression of ER stress associatedproteins activation.更多还原