【作者】 王丹萍；

【导师】 胡林森；

【作者基本信息】 吉林大学 ， 神经病学， 2010， 博士

【摘要】 为进一步探讨帕金森病(PD)的病因和病理机制,本研究在建立符合国际标准的6-OHDA诱导的SH-SY5Y细胞PD模型的基础上,采用MTT及荧光染色法检测6-OHDA对SH-SY5Y细胞的毒性作用;分别提取对照组SH-SY5Y细胞及100μM 6-OHDA处理24小时的实验组SH-SY5Y细胞的细胞总蛋白,应用荧光差异凝胶电泳(DIGE)技术获得蛋白点的差异表达信息,运用质谱(MS)技术鉴定出差异蛋白质。结果显示MTT法检测到实验组细胞存活率降低,荧光染色观察到实验组细胞凋亡的形态学改变及自噬体的数量增加,表明6-OHDA能够诱导SH-SY5Y细胞凋亡、自噬活化、促进自噬体形成最终导致细胞死亡。质谱鉴定出差异蛋白点9个,表达显著上调的有:①CCT2;②annexinA5;③Eno1 protein;④vimentin;⑤glyoxalaseⅠ;⑥Prx6;⑦14-3-3 protein。表达下调的差异蛋白质有:①Hsp90beta;②Ndufs8。在已鉴定的蛋白质中2个为蛋白质分子伴侣:CCT2和Hsp90beta;2个是与氧化应激相关的酶:glyoxalaseⅠ和Prx6;2个是与能量代谢有关的酶:Eno1 protein与Ndufs8;1个是与细胞骨架相关的蛋白质即vimentin;还有2个其它类的蛋白:14-3-3 protein和annexinA5。本研究首次应用DIGE和MALDI-TOF MS技术分析并鉴定了6-OHDA诱导的SH-SY5Y细胞PD模型中蛋白质表达水平的变化,并分别探讨了各差异表达蛋白在6-OHDA诱导的SH-SY5Y细胞PD模型中可能的作用机制。本研究发现了文献中未曾报告的CCT2、Eno1 protein、14-3-3 protein、Hsp90beta、Ndufs8、vimentin在6-OHDA诱导的SH-SY5Y细胞PD模型中的显著变化,这些发现为进一步阐明PD的发病机制提供了新的有价值线索和研究方向,可能有助于发现和发展PD临床药物治疗的新靶点并为其提供理论依据。更多还原

【Abstract】 Parkinson’s disease (PD) is a common neurodegenerative disorder whose primary pathology features are the selective degeneration and deficiency of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) and the presence of eosinophilic inclusions called Lewy body in the cytoplasm of the spared neurons. The etiology and etiopathogenesis of PD are still obscure. The death mechanism of DA neurons is not fully known so far. For a long time, researchers have been attaching importance to the hypothesis that internal and external neurotoxins induce the degeneration and death of DA neurons, and finally result in PD. As the neurotoxin similar to DA, the effect of 6-hydroxydopamine (6-OHDA) on selective central DA neurons has been extensively recognized.Finding PD key proteins and mark proteins is one of the essential problems to define the cause and precise mechanism of the disease. The technologies and methods of proteomic studies have brought new hope to this research area. On the base of traditional 2D electrophoresis technology, introduceing the inter-gel standards for the first time, combining the multiple fluorescence analysis methods, differential gel electrophoresis (DIGE) separates several different fluorescence-lablled samples on the same gel. It has become one of the most reliable and accurate technologies in quantitative and differential proteomic studies. Thus, the accuracy, reliability and repeatability have been greatly improved.To deeply explore the mechanism of PD, this study established the international-standard PD model in which 6-OHDA induces the death of SH-SY5Y cells. On the base of this PD model, this research maked use of DIGE and MALDI-TOF mass spectrometry (MS) to find the related proteins, so as to disclose the mechanism of PD at the protein level and provide new valuable clues for the early diagnosis of PD and its remedial medicines. The SH-SY5Y cells from the same batch were divided into the experimental groups and the control groups in this study. The experimental groups were treated with 6-OHDA (stock solution in ascorbic acid). The 6-OHDA final concentrations were: 25μM, 50μM, 100μM. The control groups were treated with the same amount of ascorbic acid. Cells were cultured 6h, 12h, 24h and 48h, then MTT , fluorescence staining of Hoechst 33342 and MDC were applied respectively to check the toxic effects of 6-OHDA on SH-SY5Y cells. The total proteins of the SH-SY5Y cells in the control groups and the SH-SY5Y cells treated by 100μM 6-OHDA for 24h were extracted, then the differential information from the protein points were obtained by using DIGE. In the end, the differential proteins were identified with MS technology.The viability of the cells was assayed by MTT after incubation in different-concentration of 6-OHDA for different period. The viability of SH-SY5Y cells decreased with the prolonged treatment time and the increased dose of 6-OHDA, there was obvious time and dosage dependant. This research found that 6-OHDA leads to the lower survival rates of SH-SY5Y cells. By Hoechst 33342 and MDC fluorescence staining, present study found the typical apoptosis morphological changes of the cell nucleis and the formation of autophagic vacuoles(Avs), indicating that 6-OHDA can induce apoptosis and autophagy in SH-SY5Y cells. In proteomic studies, through MS analysis and database searching, eighty-four protein spots were found differentially expressed in response to 6-OHDA administration compared with control. Nine of these were confidently identified,including: (1) molecular chaperones related proteins: heat shock 90KD protein beta subunit (Hsp90beta) , chaperonin containing TCP1, subunit2 (CCT2); (2) oxidative stress related proteins: peroxiredoxin6(Prx6), glyoxalase1; (3) cytoskeletal related protein: vimentin; (4) mitochondria related proteins :Eno1 protein (Eno1), NADH dehydrogenase (ubiquinone) Fe-S protein 8.23KD (Ndufs8); (5) other proteins: 14-3-3protein, annexin A5. The down-expressed proteins were Hsp90beta and Ndufs8, other seven proteins were up-expressed. Molecular chaperones were increased, which have protective effects on endoplasmic reticulum stress (ERS). They can promote the functional restoration of endoplasmic reticulum (ER), stabilize the calium ion level, help accurately fold, decorate and operate the accumulated proteins in ER. Moreover, they can be contributive to degrading the wrongly folded proteins so as to hold back the apoptosis of the cells. CCT2 increase indicates it is playing an important role in stabilizing cell structure and accurate protein fold. The up-regualtion of Prx6 and glyoxalase1 can increase cell’s ability to against oxidative stress and oxidative damage. Eno1 protein is one of the key enzymes in glycolytic cycle that regulates energy metabolism. The increase of Eno1 protein advances glycolytic cycle and produces ATP which provides sufficient energy to finish organismic metabolism as well as to against oxidative stress, degrade paraproteins, regulate cell transcription and cell life cycle. The down regualtion of Ndufs8 protein may prove that there is disfunction of mitochondrial respiratory chain in the model of PD induced by 6-hydroxydopamine in SH-SY5Y cells. Bisides, this research identified one protein related to apoptosis, annexin A5, which can form the high differential combination with phosphatidylserine (PS). When the apoptosis begins, PS will distribute over the surface of a cell membrane from the inner layer of the plasmolemma. The combining points between annexin A5 and cells increase, so annexin A5 is the sensitive index in the early period of apoptosis. At present, the annexin A5 fluorescence analysis has been the significant method of identifying apoptosis. Annexin A5 can inhibit the activity of phospholipase A2 and protein kinase C, take part in the activities of cystoskeleton, adjust the functions of membrane receptor, transmit mitotic signals, and promote cell secretion. The excessive expression of the annexin A5 further proves that 6-OHDA has induced apoptosis in SH-SY5Y cells.For the first time, this research has analyzed and identified the changes at the protein level in the PD model induced by 6-OHDA in SH-SY5Y cells. It has also explored differential expressing proteins’possible mechanism in the PD model induced by 6-OHDA in SH-SY5Y cells. Among the identified differential proteins, CCT2、Eno1 protein、14-3-3 protein、Hsp90beta、Ndufs8 protein、vimentin have not be found in the previous research on the PD model induced by 6-OHDA in SH-SY5Y cells. The findings of this research have supplied new valuable clues to PD mechanism and the target selection for the PD remedial medicines. These proteins might become the new diagnosis marker and the candidate target of neuroprotecive medicine treatment of PD.更多还原