MPP～+诱导的PC12细胞帕金森病模型的蛋白质组学研究
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摘要
目的研究MPP~+作用于PC12细胞48小时后蛋白质表达谱的改变，进一步从蛋白质水平上阐明帕金森病的发病机制。方法建立符合国际标准的MPP~+诱导的PC12细胞PD模型，采用MTT法检测细胞存活率，荧光染色法检测细胞凋亡百分率；提取MPP~+作用48小时后的PC12细胞总蛋白，采用差异凝胶电泳(2D-DIGE)技术分析蛋白表达谱的改变，获得蛋白点差异表达的信息；利用质谱(MALDI-TOF)技术鉴定MPP~+引起的差异表达的蛋白点。结果首次发现并鉴定了MPP~+诱导的PC12细胞PD模型中的蛋白质组学改变。MPP~+处理PC12细胞48小时后共有32个蛋白表达发生显著改变。
质谱共鉴定出7个蛋白。所鉴定的差异表达蛋白可分为5类：①具有分子伴侣活性的蛋白：新生多肽相关复合物α-多肽(nascent polypeptide associate-ed complex，α-NAC)、晶体蛋白(crystallin)，这两个蛋白的表达显著下降；②细胞骨架蛋白：神经细丝蛋白轻链多肽(neurofilament light polypeptide，NF-L)表达量显著上调；③ERM家族的埃兹蛋白(ezrin)，它的表达量显著上调；④与氧化应激、线粒体功能相关的蛋白：硫氧还原蛋白(thioredoxin，Trx)和线粒体加工肽酶(mitochondrial processing peptidases，MPPs)，这两种蛋白的表达量均显著上升；⑤免疫炎症相关的蛋白：补体结合糖蛋白(complement binder glycoprotein，gC1qBP)，表达量显著下降。这7个蛋白中除硫氧还原蛋白之外，其余均是首次用蛋白质组学方法在MPP~+诱导的PC12细胞PD模型中发现的，揭示了这些蛋白的差异改变可能与PD的发病机制相关。对这些蛋白的深入研究将使我们更好地理解PD的发病机制。这些蛋白也有可能成为新的药物作用靶点。
Part I The cytotoxicity of MPP~+ and the construction of the Parkinson's disease model
Objective: To explore the cytotoxicity of MPP~+ and construct the PD model according to the interenational standard. Methods: PC12 cells were treated with different concentrations of MPP~+(50, 100, 200, 250, 300, 500μM) for 12, 24, 48, 72, 96h, respectively. Cell viability was measured by MTT and apoptotic percentage was determined by fluorescent staining. Results: After treatment with 250μM MPP~+ for 48 hour, the PC12 cell viability decreased to 60.14% and the cell apoptotic rate increased to 9.1%. Conclusion: MPP~+ is a cytotoxic substance, which can down-regulate the cell viability and induce apoptosis. Significance: An interentional standard cell model of PD was established, which sets up a reliable basis for the study of MPP~+ mechanisms at proteomic level.
Part II Proteomic study of PC12 cell model of Parkinson's disease induced by MPP~+
Objective: To explore the mechanism of MPP~+ cytotoxicity at preteome level and find new clues for the pathogensis of PD. Method: PC12 cells were treated with or without 250μM MPP~+ for 48 hours, differential expressions of protein spots from two groups were analysed with two dimensional difference gel electrophoresis(2D-DIGE) , proteins were identified by matrix-assisted laser desorp-tion/ionization-time of flight mass spectrometer(MALDI-TOF MS). Result: About 2000 protein spots were seen in each of 2D-DIGE images. The expressions of 32 proteins were significantly changed in MPP~+ treated PC12 cells compared with the control. Among them, 15 proteins were down-regulated and 17 were up-regulated more than 30%. 7 proteins were identified by MALDI-TOF and can be classied into 5 categories:①proteins with chaperone activity : nascent polypeptide-associated complexα-polypeptide and crystallin, both of them were down-regulated;②cytoskeletal protein: neurofilament light polypeptide, it's expression was up-regulated;③the protein of ERM family, ezrin, which was also up-regulated;④oxidative stress and mitochondrial function related proteins: thioredoxin, mitochondrial processing peptidase, each of them was up-regulated;⑤immunoinflama-tion relaterd protein: complement binder glycoprotein, it's expression was down-regulated. Significance: DIGE and MS technology were used in the investigation the PC12 model of PD induced with MPP~+ for the first time and our findings provide new clues for the etiopathogenesis of PD and the candidates of therapy targets.
质谱共鉴定出7个蛋白。所鉴定的差异表达蛋白可分为5类：①具有分子伴侣活性的蛋白：新生多肽相关复合物α-多肽(nascent polypeptide associate-ed complex，α-NAC)、晶体蛋白(crystallin)，这两个蛋白的表达显著下降；②细胞骨架蛋白：神经细丝蛋白轻链多肽(neurofilament light polypeptide，NF-L)表达量显著上调；③ERM家族的埃兹蛋白(ezrin)，它的表达量显著上调；④与氧化应激、线粒体功能相关的蛋白：硫氧还原蛋白(thioredoxin，Trx)和线粒体加工肽酶(mitochondrial processing peptidases，MPPs)，这两种蛋白的表达量均显著上升；⑤免疫炎症相关的蛋白：补体结合糖蛋白(complement binder glycoprotein，gC1qBP)，表达量显著下降。这7个蛋白中除硫氧还原蛋白之外，其余均是首次用蛋白质组学方法在MPP~+诱导的PC12细胞PD模型中发现的，揭示了这些蛋白的差异改变可能与PD的发病机制相关。对这些蛋白的深入研究将使我们更好地理解PD的发病机制。这些蛋白也有可能成为新的药物作用靶点。
Part I The cytotoxicity of MPP~+ and the construction of the Parkinson's disease model
Objective: To explore the cytotoxicity of MPP~+ and construct the PD model according to the interenational standard. Methods: PC12 cells were treated with different concentrations of MPP~+(50, 100, 200, 250, 300, 500μM) for 12, 24, 48, 72, 96h, respectively. Cell viability was measured by MTT and apoptotic percentage was determined by fluorescent staining. Results: After treatment with 250μM MPP~+ for 48 hour, the PC12 cell viability decreased to 60.14% and the cell apoptotic rate increased to 9.1%. Conclusion: MPP~+ is a cytotoxic substance, which can down-regulate the cell viability and induce apoptosis. Significance: An interentional standard cell model of PD was established, which sets up a reliable basis for the study of MPP~+ mechanisms at proteomic level.
Part II Proteomic study of PC12 cell model of Parkinson's disease induced by MPP~+
Objective: To explore the mechanism of MPP~+ cytotoxicity at preteome level and find new clues for the pathogensis of PD. Method: PC12 cells were treated with or without 250μM MPP~+ for 48 hours, differential expressions of protein spots from two groups were analysed with two dimensional difference gel electrophoresis(2D-DIGE) , proteins were identified by matrix-assisted laser desorp-tion/ionization-time of flight mass spectrometer(MALDI-TOF MS). Result: About 2000 protein spots were seen in each of 2D-DIGE images. The expressions of 32 proteins were significantly changed in MPP~+ treated PC12 cells compared with the control. Among them, 15 proteins were down-regulated and 17 were up-regulated more than 30%. 7 proteins were identified by MALDI-TOF and can be classied into 5 categories:①proteins with chaperone activity : nascent polypeptide-associated complexα-polypeptide and crystallin, both of them were down-regulated;②cytoskeletal protein: neurofilament light polypeptide, it's expression was up-regulated;③the protein of ERM family, ezrin, which was also up-regulated;④oxidative stress and mitochondrial function related proteins: thioredoxin, mitochondrial processing peptidase, each of them was up-regulated;⑤immunoinflama-tion relaterd protein: complement binder glycoprotein, it's expression was down-regulated. Significance: DIGE and MS technology were used in the investigation the PC12 model of PD induced with MPP~+ for the first time and our findings provide new clues for the etiopathogenesis of PD and the candidates of therapy targets.
引文
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