【作者】 王辉；

【导师】 许予明；

【作者基本信息】 郑州大学 ， 神经病学， 2017， 硕士

【摘要】 背景帕金森病(Parkinson’s disease,PD)是仅次于阿尔兹海默病的第二大神经退行性疾病。该病以黑质致密部多巴胺能神经元严重变性、缺失和胞质内包涵体-路易小体(Lewy body,LB)生成为主要的病理特征。主要临床表现为静止性震颤、肌肉强直、运动迟缓、姿势不稳。对帕金森病死亡患者研究发现线粒体功能受损及α-突触核蛋白聚集导致氧化应激反应增加。近几十年来对PD的研究越来越受到关注,大多认为是由于环境和遗传共同作用,长期处于氧化应激状态及氧化性DNA损伤在帕金森病的发病过程中起了重要作用。1-甲基-4-苯基-1,2,3,6-吡啶(1-methyl-4-phenyl-1,2,3,-tetrahydropyridine,MPTP)能使各种哺乳动物体内纹状体和神经系统多巴胺显著减少,损伤纹状体多巴胺通路,产生与PD类似的症状。MPTP进入体内在神经胶质细胞产生的单胺氧化酶的作用下转化为它的有效成分1-甲基-4-苯基吡啶离子(1-methyl-4-phenyl-pyridine,MPP~+),后者进入线粒体抑制氧化呼吸链复合体Ⅰ酶的活性,ROS大量生成,线粒体功能障碍,造成多巴胺能神经元氧化应激损伤,在体外试验中已被广泛应用模拟PD损伤表现。然而,MPP~+诱导多巴胺能神经元死亡的机制目前尚未完全清楚,对其进行进一步的研究有助于了解PD的发病机制,寻找潜在治疗靶点。CHIP(carboxy terminus of HSP70 interacting protein)作为泛素连接酶的一种,具有E3泛素连接酶活性,在蛋白质折叠、组装、转运和降解中起着重要的调节作用。CHIP蛋白的泛素化发生在多种神经退行性疾病中,它的泛素化底物包括多种异常聚积的蛋白质,如tau,α-突触核蛋白和多聚谷氨酰胺蛋白(PolyQ)等。CHIP能与HSP70、HSP90或者其他的分子伴侣相结合,降解未折叠或者错误折叠蛋白。目前研究显示,CHIP参与维持细胞正常功能及线粒体功能,CHIP功能缺失时小鼠表现为明显的应激不耐受及迅速死亡。本研究通过过表达CHIP及抑制内源性CHIP表达CHIP-shRNA于人神经母细胞瘤细胞SH-SY5Y细胞系中,观察CHIP对MPP~+诱导的PD体外模型损伤的保护作用,并初步探讨了其可能相关的分子机制。目的1.构建MPP~+诱导的PD体外模型,筛选出MPP~+诱导SH-SY5Y细胞损伤的最适作用浓度;2.观察CHIP对MPP~+诱导的SH-SY5Y细胞损伤是否具有保护作用及其可能的分子机制;方法1.设立空白对照组,0.5,1,2,3mmol/L MPP~+浓度组,加入到SH-SY5Y细胞中,经过24小时处理后,使用CCK8法检测细胞活力,筛选出MPP~+实验处理有效浓度;2.真核细胞转染技术转染空白对照载体pcDNA-FLAG,野生型CHIP基因表达载体pcDNA-FLAG-CHIP,抑制内源性CHIP表达的pEGFP-N3-CHIP-shRNA基因表达载体,空白对照载体pEGFP-N3到人细胞母细胞瘤SH-SY5Y细胞中(分别命名为Vector组,CHIP组,Scramble组,CHIP-shRNA组)。3.实验分组:MPP~+处理组(Scramble组,CHIP-shRNA组,Vector组,CHIP组),用CCK8法检测细胞活力;通过化学发光仪检测细胞内LDH和ATP水平;通过Western Blot检测内源性线粒体凋亡相关蛋白Bax、Bcl-2的表达水平及线粒体分裂相关蛋白DRP1的表达水平。4.加入线粒体分裂相关蛋白DRP1抑制剂Mdivi-1,通过Western Blot检测DRP1的表达水平,通过化学发光仪检测细胞内LDH及ATP水平。结果1.MPP~+损伤帕金森病体外模型的建立CCK8检测结果显示,正常状态下SH-SY5Y细胞给予不同浓度的MPP~+24h后,随着药物浓度的增加,细胞存活率呈剂量依赖性降低。当MPP~+浓度维持在2mM时,细胞存活率可维持在60%左右。为使细胞处于损伤状态而又不致使细胞过度死亡,选择2mM作为后续实验浓度。2.CCK-8结果显示:与对照组相比,过表达CHIP可以减轻细胞损伤,细胞活力增加;敲除CHIP后可以加重细胞损伤,细胞活力降低(P<0.05)。3.ATP及LDH含量检测结果显示:与对照组相比,过表达CHIP可以减轻线粒体损伤,ATP含量增加,LDH释放量降低;敲除CHIP后可以加重线粒体损伤,ATP含量降低,LDH释放量增加(P<0.05)。4.Western-blot结果显示:与对照组相比,过表达CHIP可使外源性细胞凋亡相关蛋白Bax/Bcl-2比值降低;敲除CHIP后可使Bax/Bcl-2比值升高(P<0.05)。5.Western-blot结果显示:与对照组相比,过表达CHIP可使线粒体分裂相关蛋白DRP1的表达水平明显降低;敲除CHIP后可使DRP1的表达水平明显增加(P<0.05)。加入线粒体分裂蛋白DRP1抑制剂Mdivi-1,与不加入Mdivi-1的CHIP-shRNA组相比,加入Mdivi-1的CHIP-shRNA组ATP含量明显增加、LDH含量明显降低、DRP1的表达水平明显降低(P<0.05)。结论1.CHIP对MPP~+诱导的帕金森病体外模型具有保护作用,这种保护作用可能与神经细胞的线粒体功能有关。2.MPP~+诱导的细胞损伤后线粒体分裂相关蛋白DRP1的表达增多,CHIP可能是在MPP~+诱导的细胞损伤后通过抑制线粒体DRP1的表达,从而达到改善线粒体的功能发挥神经保护作用。更多还原

【Abstract】 BackgroundParkinson’s disease(PD)is the second common neurodegenerative disease after Alzheimer’s disease.The disease is characterized by severe degeneration,deletion and inclusion of Lewy body(LB)in dopaminergic neurons within dense substantia nigra.The main clinical manifestations include resting tremor,muscle rigidity,slow movement,and posture instability.Mitochondrial function damage-and α-synuclein aggregation-leaded increased oxidative stress response has been found in PD patients with death.In recent decades,the study of PD has recieved more and more attention.Due to the interaction of environment and genetic,long-term oxidative stress and oxidative DNA damage,most of them have been considered to play an important role in the pathogenesis of PD.1-methyl-4-phenyl-1,2,3-tetrahydropyridine(MPTP)can make all kinds of significant mammalian striatum and nervous system dopamine reduction,damaging the striatal dopamine pathway,further resulting in symptoms similar to PD.MPTP is converted into 1-methyl-4-phenylpyridine ion(MPP~+)by the glial cells-produced monoamine oxidase in vivo,which enters mitochondrial to inhibit the complex Ⅰ enzyme activity,ROS production,mitochondrial dysfunction of oxidative respiratory chain,resulting in dopaminergic neuronal oxidative stress injury.MPP~+ has been widely used to simulate the performance of PD damage in vitro.However,the mechanism of MPP~+-induced dopaminergic neuronal death is not yet fully understood.CHIP(Carboxy terminus of HSP70 interacting protein),a ubiquitin ligase,is a kind of auxiliary chaperone protein,which plays an important role in protein folding,assembly,transport and degradation.CHIP has E3 ubiquitin ligase activity and,can ubiquitinate tau,alpha-synuclein,and polyglutamine protein(PolyQ),which are the proteins accumulated abnormally in neurodegenerative diseases.The CHIP can bind to HSP70,HSP90 or other molecular chaperones,degrade unfolded or misfolded proteins.Current studies have shown that CHIP is involved in maintaining normal cellular function and mitochondrial function and,CHIP dysfunction is leads to stressful intolerance and rapid death.In this study,we observed the protective effect of CHIP on MPP~+-induced injury of SH-SY5 Y cells by CHIP overexpression and CHIP shRNA knockdown in SH-SY5 Y cell line of human neuroblastoma cells,and discussed the possible molecular mechanism.Objective1.To construct MPP +-induced PD in vitro model;to screen out the optimal concentration of MPP +-induced SH-SY5 Y cell injury;2.To observe the protective effect of CHIP on MPP +-induced injury of SH-SY5 Y cells and to explore the possible molecular mechanism of protection of MPP +-induced SH-SY5 Y cells induced by CHIP.Methods1.The cell viability was measured by CCK8 method,and the LDH and ATP levels were measured by luminometer.2.The pcDNA-FLAG gene expression vector pcDNA-FLAG-CHIP,the expression of pEGFP-N3-CHIP-shRNA gene expression vector in CHIP expression were transfected into human embryonic blastocysts by eukaryotic cell transfection technique(Named Vector group,CHIP group,Scramble group,CHIP-shRNA group,respectively).3.Western blot was used to detect the expression level of Bax and Bcl-2 and the expression level of mitochondrial dynamin-related protein DRP1 in mitochondria.4.Mdivi-1 was used to inhibit the activity of DRP1 protein and detect the expression levels of DRP1,the LDH and ATP levels were measured by luminometer.Results1.Establishment of MPP~+ damage SH-SY5 Y Cell Model The results of CCK8 showed that the survival rate of normal SH-SY5 Y cells decreased in a dose-dependent manner with the increase of MPP~+ concentration.When the concentration of MPP~+ was maintain at 2 mM,the cell survival rate remained at about 60%.In order to make the cells in a state of injury without causing cell death,2m M was selected as a follow-up concentration.2.CCK-8 results showed that,compared with the control group,overexpressed of CHIP could reduce cell damage and increased cell viability.The cell viability was decreased after knockout CHIP compared with the control group.3.The contents of ATP and LDH showed that overexpression of CHIP could alleviate mitochondrial damage,increase the ATP content and decrease the LDH release.After knocking out CHIP,the mitochondrial ATP content decreased and the LDH release increased.(P < 0.05)4.Western Blot showed that the expression level of Bax /Bcl-2 ratio of endogenous apoptotic protein in CHIP group was significantly higher than that in CHIP-shRNA group(P < 0.05).5.Western Blot showed that the expression level of DRP1 protein was significantly decreased by overexpressed of CHIP compared with the control group.The expression level of DRP1 protein was significantly increased after knockout CHIP.(P < 0.05)Compared with CHIP-shRNA group without Mdivi-1,the content of ATP in CHIP-shRNA group increased significantly,the content of LDH was significantly decreased and the expression level of DRP1 was significantly decreased in Mdivi-1-treated CHP-shRNA group(P <0.05)Conclusions1.CHIP has protective effects on MPP +-induced Parkinson’s disease in vitro,and this protective effect may be related to the mitochondrial function of neuronal cells.2.The expression level of DRP1 in MPP +-induced cell injury is increased,and CHIP may play a neuroprotective role in the mitochondrial function by inhibiting the expression of mitochondrial DRP1 after MPP +-induced cell injury.更多还原