本文選題：PINK1　切入點(diǎn)：突觸核蛋白　出處：《河南大學(xué)》2015年碩士論文

【摘要】：PINK1是一種線粒體絲氨酸/蘇氨酸激酶,保護(hù)細(xì)胞壓力應(yīng)激所導(dǎo)致的線粒體功能障礙,調(diào)控神經(jīng)元分化。突變的PINK1(PARK6)導(dǎo)致常染色體隱性遺傳早發(fā)性帕金森病(PD)。β-synuclein(β-syn)是α-syn同系物,其錯(cuò)義突變?chǔ)?syn P123H被發(fā)現(xiàn)存在于家族性路易體癡呆患者。突變蛋白β-syn P123H在細(xì)胞內(nèi)形成嗜酸性包涵體,并且與LAMP-2,ATP13A2,cathepsin等溶酶體標(biāo)志物共存,在胞漿聚集誘導(dǎo)細(xì)胞凋亡,具有加速神經(jīng)退化的毒性作用。PINK1能夠維持線粒體內(nèi)穩(wěn)態(tài),在PD病理學(xué)變化過程起著關(guān)鍵作用,但是PINK1通過線粒體保護(hù)作用介導(dǎo)突觸核蛋白病理學(xué)過程的機(jī)制還不清楚。本課題將在穩(wěn)定表達(dá)Parkin的He La細(xì)胞及穩(wěn)定表達(dá)β-syn P123H的B103神經(jīng)母細(xì)胞瘤細(xì)胞進(jìn)行,研究PINK1/Parkin通過調(diào)控線粒體自噬,促進(jìn)β-syn P123H自噬降解的神經(jīng)保護(hù)作用及其分子機(jī)制。本文采用PCR構(gòu)建PINK1野生型(PINK1 WT)及兩步PCR法定點(diǎn)突變技術(shù)構(gòu)建PINK1 Q126P、E240K、G309D、L347P、P498L突變真核表達(dá)載體,測序驗(yàn)證后應(yīng)用免疫印跡技術(shù)(Western Blot)鑒定其在細(xì)胞內(nèi)表達(dá)情況。WST-1細(xì)胞活性檢測及LDH細(xì)胞毒性實(shí)驗(yàn)分析PINK1 WT及突變體對細(xì)胞存活的影響。結(jié)果顯示,轉(zhuǎn)染PINK1突變體的細(xì)胞存活率下降,細(xì)胞毒性增加。相比對照組,轉(zhuǎn)染野生型PINK1質(zhì)粒的細(xì)胞無顯著性差異。應(yīng)用細(xì)胞免疫熒光化學(xué)染色的方法,觀察PINK1 WT及其突變體細(xì)胞定位情況,以及Parkin突變體T240R、R275W的細(xì)胞分布形態(tài)。運(yùn)用線粒體TMRE熒光標(biāo)記技術(shù),以FCCP組作為陽性對照,分析Parkin突變體T240R、R275W對線粒體膜電位的影響。通過脂質(zhì)體轉(zhuǎn)染及Western Blot方法,分析PINK1、Parkin對線粒體分裂融合動(dòng)態(tài)變化的影響。免疫熒光細(xì)胞化學(xué)結(jié)果顯示:PINK1突變后線粒體異常聚集。Parkin突變體在胞漿形成聚集體,并且導(dǎo)致線粒體膜電位降低。免疫印跡分析結(jié)果顯示:PINK1、Parkin的突變則導(dǎo)致了線粒體分裂、融合功能降低。野生型PINK1、Parkin參與線粒體質(zhì)量控制,促進(jìn)β-syn P123H自噬降解。免疫熒光細(xì)胞化學(xué)實(shí)驗(yàn)證明β-syn P123H及Parkin T240R聚集體經(jīng)自噬溶酶體途徑降解,Parkin T240R則加重了β-syn P123H聚集體的形成。通過自噬誘導(dǎo)劑Rapamycin,抑制劑3-MA、NH4Cl作用,結(jié)合TUNEL細(xì)胞凋亡檢測方法,研究自噬在β-syn P123H、Parkin T240R所誘導(dǎo)的細(xì)胞凋亡過程的作用。對TUNEL陽性細(xì)胞進(jìn)行計(jì)數(shù),統(tǒng)計(jì)分析結(jié)果顯示:相對于DMSO組,3-MA、NH4Cl組細(xì)胞死亡數(shù)目明顯增多,而Rapamycin組細(xì)胞死亡數(shù)目減少�？傊�,突變PINK1誘導(dǎo)線粒體斷裂形成聚集體,并且導(dǎo)致細(xì)胞活性下降,細(xì)胞毒性增加。Parkin突變體在細(xì)胞內(nèi)形成聚集體,導(dǎo)致線粒體膜電位降低。Parkin突變聚集體和β-syn P123H經(jīng)自噬溶酶體途徑降解,并且Parkin突變體加重了β-syn P123H聚集體的形成。野生型PINK1、Parkin參與線粒體質(zhì)量控制,促進(jìn)β-syn P123H自噬降解。在β-syn P123H誘導(dǎo)的突觸核蛋白病理學(xué)過程中,線粒體相關(guān)蛋白PINK1/Parkin介導(dǎo)線粒體自噬及分裂、融合過程發(fā)揮保護(hù)作用。
[Abstract]:PINK1 is a mitochondrial serine / threonine kinase that protects mitochondria from stress stress and regulates neuronal differentiation.尾 -synuclein (尾 -synn) is a homologue of 偽 -syn. The missense mutation 尾 -syn P123H has been found in familial dementia patients.The mutant 尾 -syn P123H forms eosinophilic inclusion bodies in the cells and coexists with lysosomal markers such as LAMP-2ATP 13A2cathepsin. The mutant protein 尾 -syn P123H induces apoptosis in the cytoplasm, which can accelerate neurodegenerative toxicity. PINK1 can maintain the homeostasis of mitochondria.PINK1 plays a key role in the pathological changes of PD, but the mechanism of PINK1 mediated synaptic nuclear protein pathological process through mitochondrial protection is not clear.In this study, we studied the neuroprotective effect of PINK1/Parkin on 尾 -syn P123H and 尾 -syn P123H in He-La cells and B103 neuroblastoma cells stably expressing 尾 -syn P123H by regulating mitochondrial autophagy and promoting the degradation of 尾 -syn P123H autophagy.In this paper, PCR was used to construct PINK1 wild-type PINK1 WTs and two-step PCR site-directed mutagenesis technique to construct the eukaryotic expression vector of PINK1 Q126PnE240KG309DX L347PnP498L mutation.After sequencing, Western blot technique was used to identify the expression of WST-1 in cells and the effects of PINK1 WT and mutants on cell survival were analyzed by LDH cytotoxicity test.The results showed that the survival rate and cytotoxicity of the transfected PINK1 mutants were decreased.Compared with the control group, there was no significant difference between the cells transfected with wild type PINK1 plasmid.The localization of PINK1 WT and its mutant cells and the cell distribution of Parkin mutant T240 RN R275W were observed by immunofluorescence staining.Mitochondrial TMRE fluorescence labeling technique was used to analyze the effect of Parkin mutant T240RN R275W on mitochondrial membrane potential (MMP) in FCCP group.The effects of PINK1 and Western Blot on the dynamic changes of mitochondrial mitotic fusion were analyzed by liposome transfection and Western Blot.The results of immunofluorescence cytochemistry showed that the abnormal aggregation of mitochondria. Parkin mutants formed aggregates in the cytoplasm and resulted in the decrease of mitochondrial membrane potential after 1: PINK1 mutation.Western blot analysis showed that the mutation of PINK1 Parkin resulted in mitochondrial division and decreased fusion function.Wild type PINK1 Parkin participates in mitochondrial quality control and promotes 尾 -syn P123H autophagy degradation.Immunofluorescence cytochemistry showed that 尾 -syn P123H and Parkin T240R aggregates were degraded by autophagic lysosome pathway, and the formation of 尾 -syn P123H aggregates was aggravated.The effect of autophagy on the apoptosis induced by 尾 -syn P123H ~ (2 +) Parkin T240R was studied by means of the effects of Rapamycin, an inhibitor 3-MAN NH _ 4cl, and TUNEL cell apoptosis assay.TUNEL positive cells were counted. The results of statistical analysis showed that the number of cell death in DMSO group was significantly higher than that in DMSO group, but that in Rapamycin group was decreased.In short, mutated PINK1 induced mitochondrial fragmentation to form aggregates, which led to a decrease in cellular activity and increased cytotoxicity. Parkin mutants formed aggregates in cells.The mitochondrial membrane potential decreased. Parkin mutant aggregates and 尾 -syn P123H were degraded by autophagic lysosome pathway, and the formation of 尾 -syn P123H aggregates was aggravated by Parkin mutants.Wild type PINK1 Parkin participates in mitochondrial quality control and promotes 尾 -syn P123H autophagy degradation.In the process of 尾 -syn P123H induced synaptic nuclear protein pathology, mitochondrial associated protein PINK1/Parkin mediates mitochondrial autophagy and mitosis, and the fusion process plays a protective role.
【學(xué)位授予單位】：河南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R742.5

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