Proteomic Study of a Parkinson's Disease Model of Undifferentiated SH-SY5Y Cells Induced by a Proteasome Inhibitor
Proteomic Study of a Parkinson's Disease Model of Undifferentiated SH-SY5Y Cells Induced by a Proteasome Inhibitor
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DOI：
10.7150/ijms.28595
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发表时间：
2019-01-01
影响因子：
3.6
通讯作者：
Zhang, Ying
中科院分区：
文献类型：
--
作者：
Jiang, Huiyi;Yu, Yang;Zhang, Ying
帕金森病（PD）是最常见的神经系统退行性疾病之一。然而，这种疾病的病因仍然难以捉摸。本研究建立蛋白酶体抑制剂（PSI）诱导的未分化SH-SY5Y PD模型，通过蛋白质组学研究分析其蛋白变化。方法：将培养的未分化SH-SY5Y细胞分为对照组和2.5 μ M PSI处理组（PSI处理组）。采用甲基噻唑四氮唑（MTT）法检测细胞活力。采用吖啶橙/溴化乙啶（AO/EB）、α -突触核蛋白免疫荧光、苏木精和伊红（H&E）染色分别评价细胞凋亡和胞质包涵体。分离出变化显著的蛋白点，通过2D凝胶电泳和DIGE De Cyder软件进行分析，随后通过MALDI-TOF质谱法和数据库检索进行鉴定。结果：MTT实验结果显示，PSI孵育后细胞活力有时间和剂量依赖性变化。24 h后，PSI处理组细胞早期凋亡，H&E染色和α -突触核蛋白免疫荧光检测发现PSI处理组细胞内有胞质包涵体。因此，未分化的SH-SY5Y细胞可以作为psi诱导的蛋白酶体功能抑制的PD模型。组间共差异表达18个蛋白，其中上调7个，下调11个。其中，鉴定出5个蛋白点参与泛素蛋白酶体途径诱导的PD过程。结论：线粒体热休克蛋白75 （MTHSP75）、磷酸甘油酸脱氢酶（PHGDH）、层粘连蛋白结合蛋白（LBP）、酪氨酸3/色氨酸5-单加氧酶激活蛋白（14-3-3 epsilon）和YWHAZ蛋白（14-3-3 zeta）参与线粒体功能障碍、丝氨酸合成、淀粉样蛋白清除、细胞凋亡过程和神经保护。这些发现可能为加深我们对帕金森病发病机制的认识提供新的线索。
Parkinson's disease (PD) is one of the most common nervous system degenerative diseases. However, the etiology of this disease remains elusive. Here, a proteasome inhibitor (PSI)-induced undifferentiated SH-SY5Y PD model was established to analyze protein alterations through proteomic study. Methods: Cultured undifferentiated SH-SY5Y cells were divided into a control group and a group treated with 2.5 mu M PSI (PSI-treated group). An methyl thiazolyl tetrazolium (MTT) assay was applied to detect cell viability. Acridine orange/ethidium bromide (AO/EB), alpha-synuclein immunofluorescence and hematoxylin and eosin (H&E) staining were applied to evaluate apoptosis and cytoplasmic inclusions, respectively. The protein spots that were significantly changed were separated, analyzed by 2D gel electrophoresis and DIGE De Cyder software, and subsequently identified by MALDI-TOF mass spectrometry and database searching. Results: The results of the MTT assay showed that there was a time and dose dependent change in cell viability following incubation with PSI. After 24 h incubation, PSI resulted in early apoptosis, and cytoplasmic inclusions were found in the PSI-treated group through H&E staining and alpha-synuclein immunofluorescence. Thus, undifferentiated SH-SY5Y cells could be used as PD model following PSI-induced inhibition of proteasomal function. In total, 18 proteins were differentially expressed between the groups, 7 of which were up-regulated and 11 of which were down-regulated. Among them, 5 protein spots were identified as being involved in the ubiquitin proteasome pathway-induced PD process. Conclusions: Mitochondrial heat shock protein 75 (MTHSP75), phosphoglycerate dehydrogenase (PHGDH), laminin binding protein (LBP), tyrosine 3/tryptophan 5-monooxygenase activation protein (14-3-3 epsilon) and YWHAZ protein (14-3-3 zeta) are involved in mitochondrial dysfunction, serine synthesis, amyloid clearance, apoptosis process and neuroprotection. These findings may provide new clues to deepen our understanding of PD pathogenesis.