【摘要】：背景 帕金森病（Parkinson’s disease, PD）是一種常見的神經(jīng)退行性疾病，以運(yùn)動遲緩，肌強(qiáng)直和靜止性震顫為主要臨床特征。在帕金森患者腦內(nèi)，死亡的神經(jīng)細(xì)胞多是位于炓質(zhì)區(qū)的多巴胺神經(jīng)元且多在臨床癥狀之前出現(xiàn)，，當(dāng)有明顯臨床癥狀時，大約有70%多巴胺神經(jīng)元可能已經(jīng)死亡。帕金森病的病理特征是多巴胺神經(jīng)元中α-synuclein（α-syn）包涵體的出現(xiàn)，該蛋白是路易小體（Lewy body）的主要成分。異常折疊聚集的α-syn蛋白可形成低聚物，產(chǎn)生細(xì)胞毒性，是帕金森病致病的的主要病理過程。 Numb蛋白是一種進(jìn)化保守的膜受體蛋白，在果蠅神經(jīng)系統(tǒng)研究中發(fā)現(xiàn)其具有細(xì)胞命運(yùn)決定子的作用。它具有多種蛋白質(zhì)-蛋白質(zhì)相互作用的區(qū)域，包括磷酸酪氨酸結(jié)合域（PTB）和羧基端結(jié)構(gòu)域，PTB結(jié)構(gòu)域可與泛素連接酶，如Siah1、Lnx等相互作用，對靶蛋白進(jìn)行泛素化修飾。羧基端結(jié)構(gòu)域包含一個EH結(jié)構(gòu)域結(jié)合基序和α-銜接蛋白結(jié)合位點(diǎn)，可與相關(guān)靶蛋白結(jié)合，參與調(diào)控受體蛋白胞內(nèi)循環(huán)、胞內(nèi)移行。Numb是一種內(nèi)吞蛋白，參與神經(jīng)突生長和細(xì)胞遷移過程。此外，Numb通過與Notch相互拮抗，參與調(diào)節(jié)多種生化信號途徑、調(diào)節(jié)粘附和緊密連接等諸多過程。有研究表明Numb/Notch信號通路通過調(diào)控泛素蛋白酶體系統(tǒng)，參與某些蛋白的泛素化調(diào)控。 關(guān)于Numb蛋白對α-syn蛋白寡泛素化水平的調(diào)控在國內(nèi)外尚未見相關(guān)報(bào)道，本課題將深入探求Numb蛋白是否通過參與調(diào)控α-syn蛋白寡泛素化修飾模式，參與帕金森病的致病過程。為帕金森病的早期診斷與治療提供新的思路和方法。 目的 探索Numb蛋白對α-突觸核蛋白（α-synuclein，α-syn）寡泛素化水平的調(diào)控。 方法 將EGFP-N1、EGFP-Numb分別與HA-α-syn轉(zhuǎn)染SH-SY5Y細(xì)胞；利用細(xì)胞免疫熒光法檢測α-syn蛋白和Numb蛋白的亞細(xì)胞共定位；利用免疫共沉淀的方法檢測Numb蛋白與α-syn蛋白的相互結(jié)合；利用Western blot技術(shù)檢測Numb蛋白對α-syn蛋白寡泛素水平的調(diào)控；在MPP+細(xì)胞模型中利用細(xì)胞免疫熒光檢測Numb對α-syn包涵體形成的影響。 結(jié)果 Numb與α-syn在細(xì)胞質(zhì)中存在亞細(xì)胞共定位； Numb蛋白上調(diào)α-syn寡泛素化水平； Numb蛋白通過促使α-syn寡泛素水平上調(diào)誘導(dǎo)MPP+細(xì)胞模型中α-syn陽性包涵體形成。 結(jié)論 Numb蛋白上調(diào)α-syn蛋白寡泛素水平并促進(jìn)α-syn包涵體形成。
[Abstract]:Background Parkinson's disease (Parkinson's disease, PD) is a common neurodegenerative disease characterized by motor retardation, myotonia and static tremor. In Parkinson's patients, most of the dead neurons are dopamine neurons located in the parkinsonian region and most of them appear before the clinical symptoms. When there are obvious clinical symptoms, about 70% of the dopamine neurons may have died. The pathological feature of Parkinson's disease is the presence of 偽-synuclein (偽-syn) inclusion bodies in dopamine neurons, which is the main component of Louie body (Lewy body). The abnormal folding and aggregation of 偽-syn protein can form oligomers and produce cytotoxicity, which is the main pathogenetic process of Parkinson's disease. Numb protein is an evolutionarily conserved membrane receptor protein. In the study of the nervous system of Drosophila melanogaster has been found to have the role of cell fate determinant. It has a variety of protein-protein interaction domains, including phosphotyrosine binding domain (PTB) and carboxyl terminal domain, and PTB domain can interact with ubiquitin ligases, such as Siah1,Lnx, to modify the target proteins. The carboxyl terminal domain contains a binding motif of EH domain and a binding site of 偽 -binding protein, which can bind to related target proteins and regulate the intracellular circulation of receptor proteins. Numb is an endocytosis protein. Participate in the process of neurite growth and cell migration. In addition, Numb interacts with Notch and participates in many processes, such as regulation of many biochemical signaling pathways, adhesion and tight junctions. Some studies have shown that the Numb/Notch signaling pathway participates in the regulation of ubiquitin by regulating the ubiquitin proteasome system. There have been no reports on the regulation of 偽-syn protein oligoubiquitization by Numb protein. This paper will explore whether Numb protein participates in the pathogenesis of Parkinson's disease by participating in the regulation of 偽-syn protein oligoubiquitin modification model. To provide new ideas and methods for early diagnosis and treatment of Parkinson's disease. Objective to investigate the regulation of 偽-synuclein, 偽-syn oligoubiquitin by Numb protein. Methods EGFP-N1,EGFP-Numb and HA- 偽 syn were transfected into SH-SY5Y cells, subcellular co-localization of 偽 syn protein and Numb protein was detected by immunofluorescence assay, and the interaction of Numb protein and 偽 syn protein was detected by immunoprecipitation. The effect of Numb on the formation of 偽 -syn inclusion bodies was detected by using Western blot technique, and the effect of Numb on the formation of 偽 -syn inclusion bodies in MPP cell model was detected by using Numb protein to regulate the level of 偽-syn protein oligoubiquitin. Results there were subcellular co-localization of Numb and 偽-syn in cytoplasm, Numb protein upregulated the level of 偽-syn oligobinylation. Numb protein induces the formation of 偽-syn positive inclusion bodies in MPP cells by up-regulating 偽-syn oligoubiquitin levels. Conclusion Numb protein upregulated the level of 偽-syn oligoubiquitin and promoted the formation of 偽-syn inclusion bodies.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R742.5

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1 荊婧;王雪晶;馬耀華;祝應(yīng)俊;丁曉嵐;滕軍放;;α-synuclein(A53T)蛋白調(diào)控自噬誘導(dǎo)神經(jīng)元凋亡[J];中國神經(jīng)精神疾病雜志;2013年02期

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