【摘要】：目的：神經(jīng)絲蛋白是神經(jīng)細(xì)胞重要的骨架蛋白,由NF-L、NF-M和NF-H三個(gè)亞基組成,在維持細(xì)胞骨架、穩(wěn)定細(xì)胞形態(tài)和軸突轉(zhuǎn)運(yùn)方面有十分重要作用。過(guò)度磷酸化的神經(jīng)絲是AD腦中NFT的一種主要成分和早期的病理改變,影響神經(jīng)細(xì)胞的軸突轉(zhuǎn)運(yùn)功能。糖基化是蛋白質(zhì)的另一種重要的翻譯后修飾方式。與磷酸化的性質(zhì)相似,修飾同一蛋白質(zhì)的相同或鄰近絲氨酸或者蘇氨酸的羥基,可能存在相互調(diào)節(jié)。研究表明：AD腦神經(jīng)絲蛋白的糖基化修飾水平降低,伴隨著磷酸化修飾水平增加,其機(jī)制目前尚不清楚。本實(shí)驗(yàn)在細(xì)胞水平探討神經(jīng)絲蛋白糖基化修飾水平的改變能否調(diào)節(jié)磷酸化和軸突轉(zhuǎn)運(yùn)的功能,闡明神經(jīng)絲蛋白的糖基化在AD發(fā)病機(jī)制中的作用,為AD的防治提供新的思路。方法：我們采用不同藥物通過(guò)不同機(jī)制干預(yù)SK-N-SH細(xì)胞的糖基化；采用MTT方法篩選合適的藥物濃度和對(duì)細(xì)胞活性的影響；采用蛋白免疫印跡和細(xì)胞免疫熒光化學(xué)方法觀察神經(jīng)絲蛋白糖基化和磷酸化的改變,用PI/Hoechst和DAPI染色觀察細(xì)胞的死亡方式；同時(shí),我們采用GFP-NFM轉(zhuǎn)染細(xì)胞,熒光標(biāo)記神經(jīng)絲蛋白,熒光漂白恢復(fù)技術(shù)觀察糖基化的改變對(duì)神經(jīng)絲蛋白軸突轉(zhuǎn)運(yùn)功能的影響。結(jié)果：與對(duì)照組相比,Alloxan處理細(xì)胞誘導(dǎo)細(xì)胞活性減低,神經(jīng)絲蛋白過(guò)度磷酸化且過(guò)度磷酸化的神經(jīng)絲在胞體異常聚集,而糖基化水平降低；用20μM NAG-Ae預(yù)處理12小時(shí)細(xì)胞不影響細(xì)胞的活性,提高細(xì)胞的糖基化水平,減少Alloxan誘導(dǎo)的細(xì)胞過(guò)度磷酸化和聚集；Alloxan誘導(dǎo)的細(xì)胞死亡包括程序性凋亡和繼發(fā)性壞死,而20μM NAG-Ae預(yù)處理12小時(shí)可以保護(hù)Alloxan誘導(dǎo)的細(xì)胞凋亡和壞死；Alloxan處理細(xì)胞細(xì)胞熒光漂白后恢復(fù)速度減慢,而20μM NAG-Ae處理細(xì)胞細(xì)胞熒光漂白后恢復(fù)速度增快。DON處理細(xì)胞,細(xì)胞神經(jīng)絲蛋白糖基化水平降低,過(guò)度磷酸化且過(guò)度磷酸化的神經(jīng)絲蛋白在胞體異常聚集,熒光漂白后恢復(fù)速度減慢。結(jié)論：減低細(xì)胞的糖基化能夠誘導(dǎo)AD樣神經(jīng)細(xì)胞的過(guò)度磷酸化和細(xì)胞活性下降,影響神經(jīng)絲蛋白軸突轉(zhuǎn)運(yùn)功能,而增加細(xì)胞糖基化能夠改善細(xì)胞磷酸化,有力地保護(hù)神經(jīng)細(xì)胞AD樣退行性變和神經(jīng)絲蛋白的功能。
[Abstract]:Objective: neurofilament protein is an important cytoskeleton protein of nerve cells, which is composed of three subunits of NF-L,NF-M and NF-H. It plays an important role in maintaining cytoskeleton, stabilizing cell morphology and axon transport. Hyperphosphorylated neurofilament is a major component and early pathological change of NFT in AD brain, which affects axonal transport function of nerve cells. Glycosylation is another important post-translational modification of proteins. Similar to phosphorylation, the hydroxyl groups of the same or adjacent serine or threonine modified the same protein may be interregulated. The results showed that the level of glycosylation modification of neurofilament protein in AD was decreased and the phosphorylation level was increased. The mechanism of glycosylation modification of AD was not clear. The purpose of this study was to investigate whether the changes of glycosylation and modification of neurofilament proteins can regulate phosphorylation and axon transport, and to clarify the role of neurofilament glycosylation in the pathogenesis of AD, and to provide a new idea for the prevention and treatment of AD. Methods: we used different drugs to interfere with glycosylation of SK-N-SH cells through different mechanisms, and MTT method was used to screen the appropriate concentration of drugs and their effects on cell activity. The changes of glycosylation and phosphorylation of neurofilament proteins were observed by Western blot and cellular immunofluorescence staining. The cell death patterns were observed by PI/Hoechst and DAPI staining. At the same time, we used GFP-NFM transfection, fluorescent labeling of neurofilament protein, fluorescence bleaching recovery technique to observe the effect of glycosylation on the axon transport function of neurofilament protein. Results: compared with the control group, the cell activity induced by Alloxan was decreased, and the neurofilament with excessive phosphorylation of neurofilament protein was found to have abnormal aggregation in the cell body, while the level of glycosylation was decreased. Pretreatment with 20 渭 M NAG-Ae for 12 hours did not affect cell activity, increased the level of glycosylation, and reduced Alloxan induced hyperphosphorylation and aggregation. The cell death induced by Alloxan includes programmed apoptosis and secondary necrosis, while 20 渭 M NAG-Ae pretreatment for 12 hours can protect the apoptosis and necrosis induced by Alloxan. The recovery rate of the cells treated with Alloxan was slower than that of the cells treated with 20 渭 M NAG-Ae. The level of glycosylation of neurofilament proteins in cells treated with DON was decreased, while that of the cells treated with 20 渭 M NAG-Ae was increased. The over-phosphorylated and hyperphosphorylated neurofilament proteins accumulated abnormally in the cell body, and the recovery rate was slowed down after fluorescence bleaching. Conclusion: reducing the glycosylation of cells can induce the excessive phosphorylation and decrease of cellular activity of AD like neurons, and affect the axonal transport function of neurofilament protein, but increasing the glycosylation of cells can improve the phosphorylation of cells. To protect the function of neurofilament protein and AD-like degeneration of nerve cells.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 崔冉亮;胡海燕;呂樸;戎凱;陳寧;鄧艷秋;;神經(jīng)絲蛋白質(zhì)糖基化與磷酸化的相互調(diào)節(jié)和神經(jīng)退行性疾病[J];生命的化學(xué);2009年06期

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本文編號(hào)：2394864