绿茶多酚对TDP-43相关的肌萎缩侧索硬化的细胞模型的保护作用

发布时间：2018-06-30 18:01

本文选题：肌萎缩Pt索硬化 + 运动神经元病　；参考：《河北医科大学》2014年硕士论文

【摘要】：目的：肌萎缩侧索硬化（amyotrophic lateral sclerosis，ALS）是一种进行性神经系统变性疾病，选择性侵犯脑与脊髓的上、下运动神经元，主要表现为全身进行性肌无力和肌肉萎缩。虽然近年来有关ALS的研究较多且已较为深入，但其确切病因及发病机制仍然不明。肌萎缩侧索硬化可引起死亡及长期残障，是世界范围内治愈的难题之一。肌萎缩侧索硬化给家庭社会带来了严重的经济负担。因此，遏制其发生，研究发生机制是医务工作者不可推卸的责任。最近，在ALS患者的运动神经元和胶质细胞的胞核和胞浆中均发现了异常泛素化的蛋白聚集体，主要成分是43kDa的TAR DNA结合蛋白（TDP-43)。 TAR DNA结合蛋白（TDP-43）由TARDBP基因编码，本质是一种蛋白质，具有414个氨基酸，相对分子量为43000。它包含２个ＲＮＡ识别区和一个富含甘氨酸的末端，使其可以结合单链ＤＮＡ。主要功能为转录和剪切调控。另外有研究发现，TDP-43缺失的小鼠体内存在mRNA减少、剪接错误，及一些非编码RNA。生理性TDP-43蛋白的分布主要在细胞核，少量出现在核周胞质。TDP-43蛋白表达受到许多信号如核定位信号和转移信号的调控，并易于受到机体各种生理状态的影响。在疾病和应激状态下，TDP-43蛋白可表现为一系列病理形态学改变，如核内包涵体、胞质聚集体等。TARDBP基因突变以及磷酸化、泛素化和Ｎ末端截短等修饰过程都可导致出现病理性TDP-43蛋白。与生理性结构比较，病理性TDP-43蛋白状态不稳定而更易聚集。TDP-43在ALS的病理过程中发挥重要作用，但具体机制尚不清楚。众所周知，茶多酚是茶叶中含有的一类多羟基酚类化合物的总称，其中以表没食子儿茶素没食子酸酯(EGCG)的含量最高，占绿茶多酚的25%-40%左右，，被认为是绿茶多酚生物学活性的主要来源。绿茶多酚GTP可以作为金属螯合剂和自由基清除剂，另外，还影响细胞存活和凋亡基因的过度表达以调控细胞信号转导通路、线粒体功能和泛素-蛋白酶体系统。通过这些机制，绿茶多酚对帕金森病（PD）和阿尔茨海默病（AD）等神经系统变性疾病具有防治作用。自噬是长寿命蛋白和细胞器再循环和更新的一种降解机制。许多神经变性疾病（如阿尔兹海默症，帕金森病,多聚谷氨酰氨病和ALS）都是以错误折叠的蛋白聚集为特征的。在中枢神经系统，自噬已成为了清除错误折叠蛋白的重要途径，并对神经元正常功能的维持起着重要作用；相反，下调或部分抑制自噬水平有时候会促进神经变性的发生。因此，我们研究在TDP-43转染的NSC-34的细胞的肌萎缩侧索硬化模型中，EGCG对自噬的影响，从而探讨EGCG是否对TDP-43相关的肌萎缩侧索硬化的细胞模型有保护作用。方法：本研究中使用了TDP-43转染的NSC-34的细胞作为肌萎缩侧索硬化细胞模型。用不同浓度和作用时间的绿茶多酚处理NSC34细胞后，通过CCK-8法检测其对细胞活力影响；采用恰当的浓度及时间的绿茶多酚干预NSC34细胞后，用western印迹技术检测LC3-Ⅱ蛋白水平；GFP-LC3质粒转染细胞后，用不同浓度的绿茶多酚处理该细胞，采用免疫荧光技术观察GFP-LC3点状聚集的情况；用自噬通路阻断剂Bafilomycin A1处理细胞，然后通过western印迹技术观察绿茶多酚是通过什么途径影响LC3-Ⅱ蛋白水平；在肌萎缩侧索硬化细胞模型即瞬时转染TDP-43的NSC-34的细胞中，选择恰当度的绿茶多酚干预后，用western blot法检测外源性TDP-43蛋白水平的影响。结果：（1）依次用不同浓度（0-100μM）的绿茶多酚处理NSC34细胞24h后,在0-20μΜ浓度范围的绿茶多酚对细胞活力没有明显区别，在40-80μM之间的绿茶多酚据药物浓度的逐渐增大，细胞毒性逐渐增加。在20μM的绿茶多酚处理下，处理0-36h后，细胞活力没有明显下降。（2）依次用（0-20μM）不同浓度的绿茶多酚处理NSC34细胞24h后,用western blot法检测，结果表明LC3-II的水平逐渐升高；分别用20μM的绿茶多酚处理NSC34细胞不同时间（0-36h）后, Western blot检测结果表明，LC3-II的水平在处理24h后是最高的；用免疫荧光技术观察GFP-LC3点状聚集，结果表明携带GFP-LC3点状聚集的细胞数目在处理24h内是依绿茶多酚浓度增加而逐渐增多的。（3）给予自噬通路阻断剂Bafilomycin A1阻断处理，及给予20μM的绿茶多酚处理24h后，western印迹检测结果表明，绿茶多酚在Bafilomycin A1存在的情况下，LC3-II的水平显著升高。结果表明绿茶多酚是通过自噬途径来促进蛋白的降解；在NSC34细胞中瞬时表达含有野生型、Q331K、M337V的质粒，给予20μM的绿茶多酚处理24h后，Westernblot法检测结果表明，外源性TDP-43水平都有显著的下降。结论：在TDP-43相关的ALS细胞模型中，低剂量的绿茶多酚可以上调自噬水平，促进外源性TDP-43的降解，从而发挥它的神经保护作用。
[Abstract]:Objective: amyotrophic lateral sclerosis (amyotrophic lateral sclerosis, ALS) is a progressive neurodegenerative disease that selectively invades the upper and lower motor neurons of the brain and spinal cord, mainly characterized by progressive myasthenia and atrophy of the body. Although more and more in-depth studies have been made about ALS in recent years, the exact cause and incidence of ALS have been found. The mechanism of the disease is still unknown. Amyotrophic lateral sclerosis can cause death and long-term disability. It is one of the most difficult problems in the world. Amyotrophic lateral sclerosis has brought serious economic burden to family society. Therefore, it is the responsibility of medical workers to prevent it from happening.
Recently, abnormal ubiquitination protein aggregates were found in the nucleus and cytoplasm of motor neurons and glia of ALS patients, the main component of which is the TAR DNA binding protein (TDP-43) of 43kDa.
The TAR DNA binding protein (TDP-43) is encoded by the TARDBP gene, which is essentially a protein with 414 amino acids, with a relative molecular weight of 43000. which contains 2 RNA recognition regions and a glycine rich terminal, allowing it to regulate the main function of single strand DNA. for transcriptional and shear regulation. In addition, studies have found that TDP-43 is missing. The mRNA reduction, splicing error, and the distribution of some non coded RNA. physiological TDP-43 proteins are mainly in the nucleus. A small amount of the expression of the.TDP-43 protein in the cytoplasm of the nucleus is regulated by many signals such as the nuclear location signal and the transfer signal, and is easily affected by various physiological states of the body. In the condition of disease and stress, TDP-4 3 protein can be shown as a series of pathomorphological changes, such as.TARDBP gene mutation and phosphorylation, ubiquitination and N terminal truncation, such as nuclear inclusion bodies, cytoplasmic aggregates, and N terminal truncation, which can lead to pathological TDP-43 protein. Compared with physiological structure, pathological TDP-43 egg white state is unstable and.TDP-43 is more easily aggregated in ALS disease. It plays an important role in the process of rationale, but the specific mechanism is not yet clear.
It is well known that tea polyphenols are the general name of polyhydroxy phenols in tea. The content of epigallocatechin gallate (EGCG) is the highest, accounting for about 25%-40% of green tea polyphenols, which is considered to be the main source of biological activity of green tea polyphenols. Green tea polyphenols GTP can be used as a metal chelating agent and free radical scavenging. In addition, it also affects the overexpression of cell survival and apoptosis genes to regulate cell signal transduction pathway, mitochondrial function and ubiquitin proteasome system. Through these mechanisms, green tea polyphenols have preventive effects on neurodegenerative diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD). Autophagy is a long life protein and organelle. A mechanism for degradation of recirculation and regeneration. Many neurodegenerative diseases, such as Alzheimer's, Parkinson's, polyglutamyl amidamoy and ALS, are characterized by misfolded protein aggregation. In the central nervous system, autophagy has become an important way to clear wrong folding proteins and maintain the normal function of neurons. On the contrary, down regulation or partial inhibition of autophagy can sometimes promote neurodegeneration.
Therefore, we studied the effect of EGCG on autophagy in the amyotrophic lateral sclerosis model of NSC-34 cells transfected by TDP-43, and to explore the protective effect of EGCG on the cell model of TDP-43 related amyotrophic lateral sclerosis.
Methods: in this study, TDP-43 transfected NSC-34 cells were used as amyotrophic lateral sclerosis cells. After treating NSC34 cells with green tea polyphenols with different concentration and action time, the effect on cell viability was detected by CCK-8 method. After intervention of NSC34 cells with appropriate concentration and time, Western imprinting technique was used. The level of LC3- II protein was detected by the operation. After the transfection of GFP-LC3 plasmid, the cells were treated with different concentration of green tea polyphenols. The immunofluorescence technique was used to observe the GFP-LC3 spot aggregation. The autophagy blocking agent Bafilomycin A1 was used to treat the cells. And then, through the Western blot technique, the way to observe the effect of green tea polyphenols on LC3- The level of protein II; in the amyotrophic lateral sclerosis cell model, the transient transfected TDP-43 NSC-34 cells, the appropriate degree of green tea polyphenol was selected for the prognosis, and the effect of the exogenous TDP-43 protein level was detected by Western blot method.
Results: (1) the green tea polyphenols with different concentrations (0-100 M) treated the NSC34 cell 24h in turn. The green tea polyphenols at the concentration range of 0-20 Mu had no distinct difference. The green tea polyphenols increased gradually and the cytotoxicity of the green tea polyphenols increased gradually between 40-80 Mu and M. Under the treatment of green tea polyphenols with 20 mu M, the cells survived after 0-36h treatment. The force did not decline significantly.
(2) after treating NSC34 cell 24h with different concentration of green tea polyphenols in order (0-20 M), the results showed that the level of LC3-II was gradually increased by Western blot. After treating NSC34 cells at different time (0-36h) with 20 u M green tea polyphenols, Western blot test results showed that the level of LC3-II was the highest after treating NSC34. GFP-LC3 spot aggregation was observed by optical technology. The results showed that the number of cells carrying GFP-LC3 point like aggregation was increasing in the treatment of 24h by increasing the concentration of green tea polyphenols.
(3) when the autophagy pathway blocker Bafilomycin A1 was blocked and the green tea polyphenols were treated with 20 mu M, the Western blot test showed that the green tea polyphenols increased significantly in the presence of Bafilomycin A1. The results showed that the green tea polyphenols promoted the degradation of the protein through the autophagy pathway; in the NSC34 cell, the green tea polyphenols were transient. When the plasmid containing wild type, Q331K, M337V was expressed, the green tea polyphenols were treated with 20 mu M to treat 24h. The results of Westernblot assay showed that the level of exogenous TDP-43 decreased significantly.
Conclusion: in the TDP-43 related ALS cell model, low dose of green tea polyphenols can increase the level of autophagy and promote the degradation of exogenous TDP-43, thus giving play to its neuroprotective effect.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R746.4

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