糖基化对SK-N-SH细胞神经丝的磷酸化及其功能的影响
发布时间：2018-12-29 13:04
【摘要】：目的：神经丝蛋白是神经细胞重要的骨架蛋白,由NF-L、NF-M和NF-H三个亚基组成,在维持细胞骨架、稳定细胞形态和轴突转运方面有十分重要作用。过度磷酸化的神经丝是AD脑中NFT的一种主要成分和早期的病理改变,影响神经细胞的轴突转运功能。糖基化是蛋白质的另一种重要的翻译后修饰方式。与磷酸化的性质相似,修饰同一蛋白质的相同或邻近丝氨酸或者苏氨酸的羟基,可能存在相互调节。研究表明：AD脑神经丝蛋白的糖基化修饰水平降低,伴随着磷酸化修饰水平增加,其机制目前尚不清楚。本实验在细胞水平探讨神经丝蛋白糖基化修饰水平的改变能否调节磷酸化和轴突转运的功能,阐明神经丝蛋白的糖基化在AD发病机制中的作用,为AD的防治提供新的思路。方法：我们采用不同药物通过不同机制干预SK-N-SH细胞的糖基化；采用MTT方法筛选合适的药物浓度和对细胞活性的影响；采用蛋白免疫印迹和细胞免疫荧光化学方法观察神经丝蛋白糖基化和磷酸化的改变,用PI/Hoechst和DAPI染色观察细胞的死亡方式；同时,我们采用GFP-NFM转染细胞,荧光标记神经丝蛋白,荧光漂白恢复技术观察糖基化的改变对神经丝蛋白轴突转运功能的影响。结果：与对照组相比,Alloxan处理细胞诱导细胞活性减低,神经丝蛋白过度磷酸化且过度磷酸化的神经丝在胞体异常聚集,而糖基化水平降低；用20μM NAG-Ae预处理12小时细胞不影响细胞的活性,提高细胞的糖基化水平,减少Alloxan诱导的细胞过度磷酸化和聚集；Alloxan诱导的细胞死亡包括程序性凋亡和继发性坏死,而20μM NAG-Ae预处理12小时可以保护Alloxan诱导的细胞凋亡和坏死；Alloxan处理细胞细胞荧光漂白后恢复速度减慢,而20μM NAG-Ae处理细胞细胞荧光漂白后恢复速度增快。DON处理细胞,细胞神经丝蛋白糖基化水平降低,过度磷酸化且过度磷酸化的神经丝蛋白在胞体异常聚集,荧光漂白后恢复速度减慢。结论：减低细胞的糖基化能够诱导AD样神经细胞的过度磷酸化和细胞活性下降,影响神经丝蛋白轴突转运功能,而增加细胞糖基化能够改善细胞磷酸化,有力地保护神经细胞AD样退行性变和神经丝蛋白的功能。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
本文编号：2394864
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
【参考文献】
相关期刊论文 前1条
1 崔冉亮;胡海燕;吕朴;戎凯;陈宁;邓艳秋;;神经丝蛋白质糖基化与磷酸化的相互调节和神经退行性疾病[J];生命的化学;2009年06期
，本文编号：2394864
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