【作者】 解洪荣；

【导师】 胡林森；

【作者基本信息】 吉林大学 ， 神经病学， 2010， 博士

【摘要】 本研究应用1-甲基-4-苯基-吡啶离子(MPP+)在体外构建未分化的和全反式维甲酸(ATRA)与十四烷酰佛波醇乙酸酯(TPA)序贯分化的SH-SY5Y细胞损伤的帕金森病(PD)模型,并进一步研究这两种PD细胞模型的蛋白质组学改变,筛选出与MPP+作用相关的蛋白质,为阐明PD的发病机制提供新的线索和理论依据。对构建的PD细胞模型通过MTT比色法检测细胞活性及代谢状态、Hoechst33342染色检测细胞凋亡和台盼兰染色检测细胞的死亡率。采用二维差异凝胶电泳(2D-DIGE)和基质-辅助激光解析／电离飞行时间质谱(MALDI-TOF MS)技术分析MPP+诱导的两种SH-SY5Y细胞PD模型的蛋白质组学改变。在MPP+处理24小时的未分化SH-SY5Y细胞中鉴定出3个差异表达蛋白：可溶性抗药性相关钙结合蛋白(sorcin)、膜联蛋白V (annexin V)和核糖体蛋白PO (RPLPO),它们在功能上分别与钙离子稳态、DNA损伤修复和凋亡有关。在MPP+处理48小时的ATRA和TPA序贯分化的SH-SY5Y细胞中确切地鉴定了10个差异蛋白点,代表了8个不同的蛋白质：核磷蛋白(NPM1)、含TCP1的伴侣蛋白β亚基(CCTβ)、90kDa热休克蛋白β(HSP90AB1)、14-3-3蛋白p亚型(14-3-3p,基因名Ywhab)、14-3-3蛋白ε亚型(14-3-3ε,基因名Ywhae)、14-3-3蛋白ζ亚型(14-3-3ζ,基因名Ywhaz).α-微管蛋白(a-tubulin)和3-磷酸甘油酸脱氢酶(Phgdh),他们分别参与细胞骨架的重构、分子伴侣活性、L-丝氨酸的生物合成等。这些结果表明很多途径与MPP+在SH-SY5Y细胞诱导的毒性有关,本研究结果为进一步研究PD的发病机制和合理筛选治疗靶点提供了有价值的新的线索和理论依据。在所鉴定的蛋白中,RPLPO在PD细胞模型中首次报道,NPM1、CCTβ、HSP90AB1、14-3-3β、14-3-3ε、14-3-3ζ、Phgdh和sorcin在MPP~-诱导的SH-SY5Y细胞PD模型中首次报道,膜联蛋白V表达水平上调在MPP~+诱导的SH-SY5Y细胞PD模型首次报道。更多还原

【Abstract】 Parkinson’s disease (PD) is a common progressive neurodegenerative disease pathologically characterized by the loss of dopaminergic neurons and the presence of intracellular inclusions in the spared neurons, known as Lewy bodies, in the substantia nigra pars compacta (SNpc). Despite intensive research, the exact cause of PD and the mechanisms that lead to nigro-striatal DAergic neuron death remain largely elusive. PD is considered to be resulted from complex interplay between genetic and environmental factors and mechanisms believed to contribute to the development of PD include oxidative stress, mitochondrial dysfunction, protein misfolding and aggregation, proteasome dysfunction, excitotoxicity, iron deposition and inflammation. It is likely that more than one of these mechanisms is involved in the pathogenesis of PD, yet the exact combination and succession remain unclear. In this study,2D-DIGE coupled with MALDI-ToF MS were used for identifying specific changes in protein expression in 1-methyl-4-phenyl-pyridinium (MPP+)-treated SH-SY5Y cells undifferentiated and differentiated by all-trans retinoic acid (ATRA) followed by phorbol ester 12-o-tetradecanoylphorbol-13-acetate (TPA). SH-SY5Y cells possess many characteristics of DAergic neuron, including expressing both tyrosine hydroxylase and dopamine-β-hydroxylase, as well as dopamine transporter (DAT), and when exposed to differentiation factors (RA/TPA), the cells acquire a more pronounced DAergic phenotype. Moreover, these cells can mimic many aspects of the DAergic neuron death observed in PD when treated by neurotoxins such as 1-methyl-4-phenyl-pyridinium (MPP+),6-hydroxydopamine (6-OHDA), or rotenone, so differentiated and undifferentiated SH-SY5Y cells have recently been widely used as a DAergic neuron model for PD research. MPP+ is the active metabolite of the dopamine specific neurotoxin 1-methyl-4-phenyl-1.2,3,6-tetrahydropyridine (MPTP), which has been widely used to induce experimental PD model for more than 20 years. Thus MPP+-induced SH-SY5Y cells were chosen to study the pathogenesis of PD in this study. Proteomics represents a powerful tool to perform high-throughput studies at protein level, allowing the detection of the appearance of new proteins, differences in the amount of expressed proteins, and changes in post-translational modified (PTM)。In this study, comprehensive analysis of high-throughout protein data was done on experimental PD mimics by proteomics research, which unquestionably contribute to comprehensive recognition of PD.Objective:To observe the different sensitivity of undifferentiated, ATRA-differentiated and ATRA/TPA-differentiated SH-SY5Y cells to 1-methyl-4-phenyl-pyridinium ion (MPP+), a kind of PD mimicking neurotoxin, in order to make better use of SH-SY5Y cells in neurotoxicity and PD research. Then to built in vitro PD cell models in undifferentiated SH-SY5Y cells treated with 1.0 mM MPP+for 24 h and ATRA/TPA-differentiated SH-SY5Y cells treated with 1.0 mM MPP+for 48 h. And to identify the differentially expressed proteins in these two kinds of PD cell models for revealing the potential pathogenesis of PD.Methods:SH-SY5Y cells were differentiated with 10μM ATRA for 6 days or 10μM ATRA for 3 days followed by 80 nM TPA for another 3 days (RA/TPA) and undifferentiated SH-SY5Y cells were cultured at the same time and condition. Then each of these groups was treated with different concentration of MPP+(0.05,0.1,0.25,0.5,1.0 and 2.0 mM) for different time (24,48, and 72 h). Cell morphology was observed under inverse microscopy, cell viability was calculated by the method of MTT assay and trypan blue exclusion was employed to detect the death rate of three groups of SH-SY5Y cells treated with 1mM MPP+ for 48 h. Then, we built in vitro PD cell models in undifferentiated SH-SY5Y cells treated with MPP+ for 24 h and ATRA/TPA-differentiated SH-SY5Y cells treated with MPP+ for 48 h. Cell apoptosis was observed via Hoechst 33342 staining, and cell viability was calculated by MTT assay and trypan blue exclusion. Quantitative two-dimensional difference in-gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to determine the changing protein levels in the two kinds of PD cell models.Results:ATRA and ATRA/TPA could induce morphological differentiation, inhibit growth of SH-SY5Y cells and showed time-and dose-dependent manner. After treated with MPP+, the cell viability declined in a time-and concentration-dependent manner in three groups of SH-SY5Y cells. Trypan blue exclusion and MTT assay showed that SH-SY5Y cells with different differentiation status have different sensitivity to MPP+. The results showed that ATRA/TPA-differentiated SH-SY5Y cells is the most sensitive cells to MPP+, the next is undifferentiated SH-SY5Y cells, and ATRA-differentiated SH-SY5Y cells are the least sensitive cells. In undifferentiated SH-SY5Y cells treated with 1.0 mM MPP+for 24 h, the cell viability decreased to 75.8%±1.5%, apoptosis rate reached to 23%±2.5%, and the death rate was 25.7%±1.5%. And in ATRA/TPA-differentiated SH-SY5Y cells treated with 1.0 mM MPP+for 48 h, the cell viability decreased to 54.3%±0.8%, apoptosis rate reached to 35.3%±1.5%, and the death rate was 38.3%±2.1%.After MPP+-induced undifferentiated and ATRA/TPA-differentiated SH-SY5Y cells PD models were built, a comparative proteomic approach (2D-DIGE coupled with MALDI-ToF MS) was performed to identify differentially expressed proteins in PD models. Via comparing MPP+-treated undifferentiated SH-SY5Y cells to controls, a total of 22 proteins displayed at least a 1.3-fold change in relative abundance. Among with, three proteins were identified successfully as sorcin (soluble resistance-related calcium binding protein), annexin V (annexin A5, ANXA5), and ribosomal protein P0 (ribosomal phosphoprotein P0, RPLPO), which in function are associated respectively with apoptosis, calcium homeostasis, and DNA insults. Via comparing MPP+-treated ATRA/TPA-differentiated SH-SY5Y cells to controls,49 protein spots were found to be differentially expressed. Among which,10 protein spots were unambiguously identified, representing 8 distinct proteins:nucleophosmin (NPM1), chaperonin containing TCP 1 subunitβ(CCTβ), heat shock 90 kDa protein 1, beta (HSP90AB1 or HSP90-β),14-3-3 beta (14-3-3β, gene symbol Ywhab),14-3-3 epsilon (14-3-3ε, gene symbol Ywhae)、14-3-3 zeta (14-3-3ζ,, gene symbol Ywhaz),α-tubulin, 3-phosphoglcerate dehydrogenase (Phgdh). In some cases, more than one protein spots corresponding to the same protein, which might represent different PTM forms of the same protein, leading to minor drift of molecular weight and isoelectric point. The function of these proteins have implication on pathways such as cytoskeletal reorgnization, chaperone activity, and L-serine biosythesis, and the upregulation of these chaperone proteins in present study provide further evidence for a role of ER stress and UPR in pathogenesis of PD.Conclusion:These results indicated that multiple mechanisms may be pertinent in the underlying pathogenesis of PD, and provided new valuable clues for the further exploration of the pathogenesis and treatment of PD.更多还原