本文選題：肌萎縮Pt索硬化 + 運(yùn)動(dòng)神經(jīng)元病　； 參考：《河北醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：肌萎縮側(cè)索硬化（amyotrophic lateral sclerosis，ALS）是一種進(jìn)行性神經(jīng)系統(tǒng)變性疾病，選擇性侵犯腦與脊髓的上、下運(yùn)動(dòng)神經(jīng)元，主要表現(xiàn)為全身進(jìn)行性肌無(wú)力和肌肉萎縮。雖然近年來(lái)有關(guān)ALS的研究較多且已較為深入，但其確切病因及發(fā)病機(jī)制仍然不明。肌萎縮側(cè)索硬化可引起死亡及長(zhǎng)期殘障，是世界范圍內(nèi)治愈的難題之一。肌萎縮側(cè)索硬化給家庭社會(huì)帶來(lái)了嚴(yán)重的經(jīng)濟(jì)負(fù)擔(dān)。因此，遏制其發(fā)生，研究發(fā)生機(jī)制是醫(yī)務(wù)工作者不可推卸的責(zé)任。 最近，在ALS患者的運(yùn)動(dòng)神經(jīng)元和膠質(zhì)細(xì)胞的胞核和胞漿中均發(fā)現(xiàn)了異常泛素化的蛋白聚集體，主要成分是43kDa的TAR DNA結(jié)合蛋白（TDP-43)。 TAR DNA結(jié)合蛋白（TDP-43）由TARDBP基因編碼，本質(zhì)是一種蛋白質(zhì)，具有414個(gè)氨基酸，相對(duì)分子量為43000。它包含２個(gè)ＲＮＡ識(shí)別區(qū)和一個(gè)富含甘氨酸的末端，使其可以結(jié)合單鏈ＤＮＡ。主要功能為轉(zhuǎn)錄和剪切調(diào)控。另外有研究發(fā)現(xiàn)，TDP-43缺失的小鼠體內(nèi)存在mRNA減少、剪接錯(cuò)誤，及一些非編碼RNA。生理性TDP-43蛋白的分布主要在細(xì)胞核，少量出現(xiàn)在核周胞質(zhì)。TDP-43蛋白表達(dá)受到許多信號(hào)如核定位信號(hào)和轉(zhuǎn)移信號(hào)的調(diào)控，并易于受到機(jī)體各種生理狀態(tài)的影響。在疾病和應(yīng)激狀態(tài)下，TDP-43蛋白可表現(xiàn)為一系列病理形態(tài)學(xué)改變，如核內(nèi)包涵體、胞質(zhì)聚集體等。TARDBP基因突變以及磷酸化、泛素化和Ｎ末端截短等修飾過(guò)程都可導(dǎo)致出現(xiàn)病理性TDP-43蛋白。與生理性結(jié)構(gòu)比較，病理性TDP-43蛋白狀態(tài)不穩(wěn)定而更易聚集。TDP-43在ALS的病理過(guò)程中發(fā)揮重要作用，但具體機(jī)制尚不清楚。 眾所周知，茶多酚是茶葉中含有的一類(lèi)多羥基酚類(lèi)化合物的總稱(chēng)，其中以表沒(méi)食子兒茶素沒(méi)食子酸酯(EGCG)的含量最高，占綠茶多酚的25%-40%左右，，被認(rèn)為是綠茶多酚生物學(xué)活性的主要來(lái)源。綠茶多酚GTP可以作為金屬螯合劑和自由基清除劑，另外，還影響細(xì)胞存活和凋亡基因的過(guò)度表達(dá)以調(diào)控細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)通路、線粒體功能和泛素-蛋白酶體系統(tǒng)。通過(guò)這些機(jī)制，綠茶多酚對(duì)帕金森�。≒D）和阿爾茨海默�。ˋD）等神經(jīng)系統(tǒng)變性疾病具有防治作用。自噬是長(zhǎng)壽命蛋白和細(xì)胞器再循環(huán)和更新的一種降解機(jī)制。許多神經(jīng)變性疾�。ㄈ绨柶澓ＤY，帕金森病,多聚谷氨酰氨病和ALS）都是以錯(cuò)誤折疊的蛋白聚集為特征的。在中樞神經(jīng)系統(tǒng)，自噬已成為了清除錯(cuò)誤折疊蛋白的重要途徑，并對(duì)神經(jīng)元正常功能的維持起著重要作用；相反，下調(diào)或部分抑制自噬水平有時(shí)候會(huì)促進(jìn)神經(jīng)變性的發(fā)生。 因此，我們研究在TDP-43轉(zhuǎn)染的NSC-34的細(xì)胞的肌萎縮側(cè)索硬化模型中，EGCG對(duì)自噬的影響，從而探討EGCG是否對(duì)TDP-43相關(guān)的肌萎縮側(cè)索硬化的細(xì)胞模型有保護(hù)作用。 方法：本研究中使用了TDP-43轉(zhuǎn)染的NSC-34的細(xì)胞作為肌萎縮側(cè)索硬化細(xì)胞模型。用不同濃度和作用時(shí)間的綠茶多酚處理NSC34細(xì)胞后，通過(guò)CCK-8法檢測(cè)其對(duì)細(xì)胞活力影響；采用恰當(dāng)?shù)臐舛燃皶r(shí)間的綠茶多酚干預(yù)NSC34細(xì)胞后，用western印跡技術(shù)檢測(cè)LC3-Ⅱ蛋白水平；GFP-LC3質(zhì)粒轉(zhuǎn)染細(xì)胞后，用不同濃度的綠茶多酚處理該細(xì)胞，采用免疫熒光技術(shù)觀察GFP-LC3點(diǎn)狀聚集的情況；用自噬通路阻斷劑Bafilomycin A1處理細(xì)胞，然后通過(guò)western印跡技術(shù)觀察綠茶多酚是通過(guò)什么途徑影響LC3-Ⅱ蛋白水平；在肌萎縮側(cè)索硬化細(xì)胞模型即瞬時(shí)轉(zhuǎn)染TDP-43的NSC-34的細(xì)胞中，選擇恰當(dāng)度的綠茶多酚干預(yù)后，用western blot法檢測(cè)外源性TDP-43蛋白水平的影響。 結(jié)果：（1）依次用不同濃度（0-100μM）的綠茶多酚處理NSC34細(xì)胞24h后,在0-20μΜ濃度范圍的綠茶多酚對(duì)細(xì)胞活力沒(méi)有明顯區(qū)別，在40-80μM之間的綠茶多酚據(jù)藥物濃度的逐漸增大，細(xì)胞毒性逐漸增加。在20μM的綠茶多酚處理下，處理0-36h后，細(xì)胞活力沒(méi)有明顯下降。 （2）依次用（0-20μM）不同濃度的綠茶多酚處理NSC34細(xì)胞24h后,用western blot法檢測(cè)，結(jié)果表明LC3-II的水平逐漸升高；分別用20μM的綠茶多酚處理NSC34細(xì)胞不同時(shí)間（0-36h）后, Western blot檢測(cè)結(jié)果表明，LC3-II的水平在處理24h后是最高的；用免疫熒光技術(shù)觀察GFP-LC3點(diǎn)狀聚集，結(jié)果表明攜帶GFP-LC3點(diǎn)狀聚集的細(xì)胞數(shù)目在處理24h內(nèi)是依綠茶多酚濃度增加而逐漸增多的。 （3）給予自噬通路阻斷劑Bafilomycin A1阻斷處理，及給予20μM的綠茶多酚處理24h后，western印跡檢測(cè)結(jié)果表明，綠茶多酚在Bafilomycin A1存在的情況下，LC3-II的水平顯著升高。結(jié)果表明綠茶多酚是通過(guò)自噬途徑來(lái)促進(jìn)蛋白的降解；在NSC34細(xì)胞中瞬時(shí)表達(dá)含有野生型、Q331K、M337V的質(zhì)粒，給予20μM的綠茶多酚處理24h后，Westernblot法檢測(cè)結(jié)果表明，外源性TDP-43水平都有顯著的下降。 結(jié)論：在TDP-43相關(guān)的ALS細(xì)胞模型中，低劑量的綠茶多酚可以上調(diào)自噬水平，促進(jìn)外源性TDP-43的降解，從而發(fā)揮它的神經(jīng)保護(hù)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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R746.4

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相關(guān)期刊論文 前1條

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