發(fā)布時間：2018-07-29 08:07

【摘要】：背景:肌萎縮側(cè)索硬化[Amyotrophic lateral sclerosis,(ALS)],是一種致死性的運(yùn)動神經(jīng)元變性疾病,引起漸進(jìn)性肌肉無力、癱瘓和過早死亡。同時ALS是一種多因素疾病,有病因?qū)W異質(zhì)性和高度可變的臨床表現(xiàn)。它的特征是選擇性上下運(yùn)動神經(jīng)元變性和死亡,中年起病,在1-5年漸進(jìn)性癱瘓和死亡。最近,在肌萎縮側(cè)索硬化和伴有泛素陽性包涵體的額顳葉癡呆患者[frontotemporal lobar dementia with ubiquitin-positive inclusions,(FTLD-U)]以及帕金森氏病、路易體癡呆及30%的阿爾茨海默氏病患者的腦組織中發(fā)現(xiàn)了TARDNA結(jié)合蛋白43 k Da(TDP-43)陽性包涵體。TDP-43是一種保守的,廣泛表達(dá)的核蛋白,由1號染色體上的TARDBP基因編碼。在結(jié)構(gòu)上有5個功能區(qū)域,包括2個RNA識別模體(RRM1和RRM2),一個富含甘氨酸區(qū)域(GRR),一個核定位信號(NLS)和細(xì)胞核輸出信號(NES)來介導(dǎo)核與胞質(zhì)的穿梭運(yùn)動。功能上與外顯子跳躍和選擇性剪接有關(guān)。病理性TDP43從核再分布到細(xì)胞質(zhì),在胞質(zhì)聚集。自從2006年在散發(fā)的ALS患者和FTLD患者中發(fā)現(xiàn)以TDP-43作為主要組分的泛素蛋白聚集體,為我們理解ALS的發(fā)病機(jī)制補(bǔ)充了一個主要的視野。隨后的研究發(fā)現(xiàn),散發(fā)性和家族性FTLD-U和ALS患者以胞質(zhì)內(nèi)聚集不溶性的、過度磷酸化的、泛素化的和蛋白水解裂開的C端片段在受累的腦和脊髓區(qū)域堆積為特征。尤其是25kd的TDP-43 C末端片段(TDP-25)在受累的腦區(qū)域聚集提示它可能涉及疾病的發(fā)病機(jī)制。TDP-43在RNA代謝、神經(jīng)突起增長、神經(jīng)元發(fā)育以及應(yīng)激顆粒組成中起重要作用。研究表明TDP-25過度表達(dá)足以引起TDP-43的錯誤定位和胞質(zhì)內(nèi)堆積內(nèi)源性全長TDP-43。但是在ALS相關(guān)的細(xì)胞模型中,仍未建立穩(wěn)定轉(zhuǎn)染TDP-25的細(xì)胞系。ALS的一個病理特點為:在運(yùn)動神經(jīng)元內(nèi)有異常的聚集體,而且TDP-43包涵體中富含TDP-43的C末端片段。在真核細(xì)胞中,關(guān)于蛋白質(zhì)的清除主要有兩個系統(tǒng):泛素蛋白酶體系統(tǒng)和自噬-溶酶體系統(tǒng)。泛素蛋白酶體系統(tǒng)在維持細(xì)胞蛋白質(zhì)平衡上起重要作用,參與多個細(xì)胞過程,比如細(xì)胞周期、細(xì)胞的分化和發(fā)展、應(yīng)激反應(yīng)和DNA修飾。許多神經(jīng)變性病在腦組織形成泛素化的包涵體就是通過這個途徑來降解的。TDP-43位于腦組織的泛素陽性包涵體內(nèi),表明泛素蛋白酶體系統(tǒng)可能參與TDP-43的降解或它的發(fā)病機(jī)制。由于化學(xué)結(jié)構(gòu)的不同,蛋白酶體抑制劑大致可以分為兩類,天然產(chǎn)物和合成類似物。肽醛類抑制劑是被確定的第一種蛋白酶體抑制劑,由于合成和優(yōu)化的方便研究人員已經(jīng)開發(fā)出了很多種,他們是使用最廣泛的抑制劑。1994年由Rock研制出的MG132(Z-Leu-Leu-Leu-al,also termed Cbz-LLL or z-LLL)是第一種被確定的而且是經(jīng)典的蛋白酶體抑制劑,在蛋白酶體生物學(xué)上被廣泛應(yīng)用。MG132是一種可逆的、有效的26S肽醛類抑制劑,它可以抑制蛋白酶體通路上蛋白質(zhì)的降解,進(jìn)而影響細(xì)胞的增殖,促進(jìn)細(xì)胞凋亡。在天然抑制劑中,乳胞素(Lactacystin)是眾所周知的、細(xì)胞可滲透的蛋白酶體抑制劑。它是存在于土壤中的鏈霉菌屬的代謝物,是一種選擇性20S蛋白酶體的抑制劑。乳胞素與其活性中間體的β2內(nèi)酯可以選擇性地、不可逆地結(jié)合于哺乳動物的蛋白酶體的β5亞單位,進(jìn)而抑制多種肽酶的活性。自噬-溶酶體系統(tǒng)參與清除疾病蛋白以及除掉聚集的蛋白質(zhì)或損傷的細(xì)胞器如線粒體。在神經(jīng)變性病,易于聚集的疾病蛋白質(zhì)通常對于蛋白酶體的降解有抵抗,更傾向于通過自噬-溶酶體系統(tǒng)降解。3MA(3-Methyladenine),是一種被廣泛使用的自噬抑制劑,通過抑制PI3K阻斷自噬體的形成來抑制自噬。巴弗洛霉素A1(Bafilomycin A1)是一種來源于灰色鏈霉素的囊泡性H+-ATP酶質(zhì)子泵抑制劑,屬于大環(huán)內(nèi)酯類抗生素,它可以抑制囊泡酸化從而抑制自噬體后期的形成。雷帕霉素(Rapamycin)是一種分離于放線菌培養(yǎng)液中的新型大環(huán)內(nèi)酯類抗生素。它通過形成FK506結(jié)合蛋白(FKBP12)的復(fù)合物抑制雷帕霉素靶蛋白(mammalian target of rapamycin,m TOR)的激活,從而導(dǎo)致自噬激活。以往的研究表明TDP-43可以通過泛素-蛋白酶體途徑和自噬-溶酶體途徑降解,但是對于穩(wěn)定轉(zhuǎn)染TDP-25細(xì)胞系中聚集體的降解以及是否有毒性仍未見報道。第一部分穩(wěn)轉(zhuǎn)TDP-25細(xì)胞模型的建立及鑒定目的:本研究的目的是建立穩(wěn)定轉(zhuǎn)染TDP-25的細(xì)胞模型,然后鑒定是否成功。方法:按照操作流程,應(yīng)用脂質(zhì)體轉(zhuǎn)染的方法在NSC34細(xì)胞中瞬時轉(zhuǎn)染empty p CI-neo質(zhì)粒和TDP-25c DNAs質(zhì)粒。在細(xì)胞轉(zhuǎn)染質(zhì)粒48 h后,將培養(yǎng)液更換為含有G418的培養(yǎng)液培養(yǎng),3周后挑選抵抗G418的表達(dá)質(zhì)粒的多克隆細(xì)胞。用流式細(xì)胞儀分選技術(shù)分離出轉(zhuǎn)染質(zhì)粒的細(xì)胞,即帶有綠色熒光標(biāo)簽的空質(zhì)粒細(xì)胞以及TDP-25細(xì)胞。培養(yǎng)分選出的表達(dá)TDP-25的多克隆細(xì)胞,收集生長狀態(tài)良好的細(xì)胞并用Western印跡方法驗證細(xì)胞中質(zhì)粒的表達(dá);用倒置熒光顯微鏡及激光共聚焦顯微鏡觀察TDP-25蛋白的分布特點;使用透射電子顯微鏡觀察穩(wěn)轉(zhuǎn)TDP-25細(xì)胞中聚集體的形態(tài);MDA方法檢測穩(wěn)轉(zhuǎn)細(xì)胞系的脂質(zhì)過氧化作用;用CCK-8技術(shù)檢測TDP-25細(xì)胞的活力;Mito Tracker#174;Red CM-H2XRos檢測活性氧;WB方法檢測凋亡標(biāo)志物的表達(dá)來反應(yīng)穩(wěn)轉(zhuǎn)細(xì)胞系的毒性作用。結(jié)果:(1)通過流式細(xì)胞儀分選技術(shù)可以獲得穩(wěn)定轉(zhuǎn)染的TDP-25細(xì)胞系和空質(zhì)粒細(xì)胞系。Western印跡數(shù)據(jù)顯示:TDP-25的表達(dá)水平大約是內(nèi)源性TDP-43的47%。(2)在穩(wěn)轉(zhuǎn)的細(xì)胞系中,TDP-25蛋白既位于細(xì)胞核中,也可位于細(xì)胞漿中。給予蛋白酶體抑制劑MG132后TDP-25在胞核和胞漿更容易形成致密的聚集體。透射電子顯微鏡下觀察聚集體為纖維束狀的電子致密顆粒。(3)穩(wěn)定轉(zhuǎn)染TDP-25的細(xì)胞存在氧化應(yīng)激、脂質(zhì)過氧化和細(xì)胞凋亡。(4)通過激光共聚焦顯微鏡觀察,在TDP-25穩(wěn)轉(zhuǎn)細(xì)胞中,聚集體可以與自噬相關(guān)蛋白P62以及泛素進(jìn)行共定位。結(jié)論:通過流式細(xì)胞儀分選技術(shù)建立了穩(wěn)轉(zhuǎn)細(xì)胞系。和對照細(xì)胞相比,穩(wěn)定轉(zhuǎn)染TDP-25的細(xì)胞可以引起氧化應(yīng)激、脂質(zhì)過氧化和凋亡。第二部分泛素-蛋白酶體系統(tǒng)對穩(wěn)轉(zhuǎn)TDP-25細(xì)胞的影響目的:本研究的目的是探討泛素-蛋白酶體系統(tǒng)對穩(wěn)定轉(zhuǎn)染TDP-25細(xì)胞中聚集體的影響以及TDP-25細(xì)胞是否存在毒性。方法:使用通過流式細(xì)胞儀分選技術(shù)建立的穩(wěn)轉(zhuǎn)細(xì)胞系,將細(xì)胞傳代至六孔板中,分別給予蛋白酶體抑制劑MG132或乳胞素后觀察聚集體是否通過泛素-蛋白酶體通路進(jìn)行降解。在分別干預(yù)3、6、12、24小時后,使用倒置熒光顯微鏡觀察TDP-25聚集體或GFP熒光數(shù)量的變化,并收集細(xì)胞使用Western印跡方法對TDP-25蛋白質(zhì)的表達(dá)進(jìn)行定量測定。使用透射電子顯微鏡觀察穩(wěn)轉(zhuǎn)細(xì)胞系的線粒體的情況。結(jié)果:(1)在穩(wěn)轉(zhuǎn)TDP-25細(xì)胞中,TDP-25蛋白質(zhì)的表達(dá)呈時間依賴性逐漸增加,在蛋白酶體抑制劑干預(yù)12小時后達(dá)到高峰。然而,內(nèi)源性TDP-43和空質(zhì)粒對照組沒有發(fā)現(xiàn)改變。(2)在穩(wěn)轉(zhuǎn)細(xì)胞中TDP-25的熒光數(shù)量呈時間依賴性增加,在蛋白酶體抑制劑干預(yù)24小時達(dá)到3000。然而空質(zhì)粒組的熒光沒有明顯改變。(3)當(dāng)TDP-25過表達(dá)時,它具有易于聚集的性質(zhì)。在穩(wěn)定轉(zhuǎn)染TDP-25細(xì)胞系,我們發(fā)現(xiàn)TDP-25在核膜附近形成一些小的點狀的聚集體。然而,經(jīng)蛋白酶體抑制劑MG132干預(yù)后,TDP-25在細(xì)胞質(zhì)和細(xì)胞核形成更大的聚集體。(4)穩(wěn)轉(zhuǎn)TDP-25細(xì)胞中的線粒體是腫脹的,線粒體嵴是擴(kuò)張的。給予蛋白酶體抑制劑后,線粒體的這種改變更加明顯。結(jié)論:穩(wěn)定轉(zhuǎn)染TDP-25細(xì)胞系中聚集體的降解主要是通過泛素-蛋白酶體系統(tǒng),其線粒體是異常的。這個細(xì)胞系的毒性依賴于蛋白酶體的活性。第三部分自噬-溶酶體系統(tǒng)對穩(wěn)轉(zhuǎn)TDP-25細(xì)胞的影響目的:本研究的目的是探討自噬-溶酶體系統(tǒng)對穩(wěn)定表達(dá)TDP-25細(xì)胞的影響。方法:使用通過流式細(xì)胞儀分選技術(shù)建立的穩(wěn)轉(zhuǎn)細(xì)胞系,將生長狀態(tài)良好的細(xì)胞傳代至六孔板中,分別選用不同濃度的自噬通路阻斷劑(巴弗洛霉素A1和3MA)和自噬通路激活劑(雷帕霉素)處理細(xì)胞,觀察穩(wěn)轉(zhuǎn)細(xì)胞中TDP-25聚集體是否通過自噬通路降解。使用倒置熒光顯微鏡觀察聚集體熒光數(shù)量的變化,并收集細(xì)胞使用Western印跡方法對TDP-25蛋白的表達(dá)進(jìn)行定量測定。結(jié)果:(1)分別使用不同濃度的自噬途徑抑制劑巴弗洛霉素A1和3MA干預(yù)穩(wěn)轉(zhuǎn)細(xì)胞24 h后,倒置熒光顯微鏡及Western印跡方法均未檢測到聚集體形態(tài)、位置及數(shù)量的改變。(2)分別使用不同濃度的自噬途徑激活劑雷帕霉素處理穩(wěn)轉(zhuǎn)細(xì)胞24h后,倒置熒光顯微鏡及Western印跡方法仍未檢測到聚集體形態(tài)、位置及數(shù)量的改變。結(jié)論:穩(wěn)定轉(zhuǎn)染TDP-25細(xì)胞系中聚集體的降解可能不通過自噬-溶酶體系統(tǒng)。
[Abstract]:Background: amyotrophic lateral sclerosis, [Amyotrophic lateral sclerosis, (ALS)), is a fatal motor neuron degeneration disease that causes progressive muscle weakness, paralysis, and premature death. ALS is a multifactor disease with a heterogeneous and highly variable clinical manifestation of etiology. It is characterized by selective upper and lower motor neuron change. Sexual and death, middle-aged onset, progressive paralysis and death in 1-5 years. Recently, [frontotemporal lobar dementia with ubiquitin-positive inclusions, (FTLD-U)) in amyotrophic lateral sclerosis and ubiquitin positive inclusion bodies, as well as Parkinson's disease, Louis's dementia and the brain of 30% Alzheimer's disease patients It was found that the TARDNA binding protein 43 K Da (TDP-43) positive inclusion body.TDP-43 was a conservative, widely expressed nucleoprotein, encoded by the TARDBP gene on chromosome 1. There were 5 functional regions in the structure, including 2 RNA recognition modules (RRM1 and RRM2), a glycine region (GRR), a nuclear localization signal (NLS) and cells. The nuclear output signal (NES) mediates the shuttle movement of the nucleus and cytoplasm. Function is related to exon hopping and selective splicing. Pathological TDP43 is redistributed from the nucleus to the cytoplasm and in the cytoplasm. Since 2006, the ubiquitin protein aggregates with TDP-43 as the main component in the sporadic ALS patients and FTLD patients are found to understand the hair of ALS. The pathogenesis supplemented a major field of vision. Subsequent studies have found that sporadic and familial FTLD-U and ALS patients accumulate insoluble, overphosphorylated, ubiquitinated and proteolytic cleaved C terminal fragments in the affected brain and spinal regions, especially the TDP-43 C terminal fragment of 25kd (TDP-25) involved in the involvement of the disease. Regional aggregation of the brain suggests that it may involve the pathogenesis of disease,.TDP-43, which plays an important role in RNA metabolism, neurite growth, neuron development, and stress particle composition. The study shows that overexpression of TDP-25 is sufficient to cause the wrong localization of TDP-43 and the accumulation of endogenous TDP-43. in the cytoplasm, but in the ALS related cell model, it is still still in the cell model. A pathological feature of the cell line.ALS without stable transfection of TDP-25 is that there are abnormal aggregates in the motor neuron and the C terminal fragment of the TDP-43 in the TDP-43 inclusion body. In eukaryotic cells, there are two main systems for the removal of protein: the ubiquitin proteasome system and autophagy lysosome system. Ubiquitin proteasome The system plays an important role in maintaining cell protein balance. It participates in multiple cell processes, such as cell cycle, cell differentiation and development, stress response and DNA modification. The ubiquitin inclusion bodies of many neurodegenerative diseases in the brain tissue are the ubiquitin positive inclusion bodies of.TDP-43 in the brain tissue that are degraded through this pathway. The proteasome system may be involved in the degradation of TDP-43 or its pathogenesis. Due to different chemical structures, proteasome inhibitors can be roughly divided into two types, natural products and synthetic analogues. The peptide inhibitor is the first proteasome inhibitor, which has been developed by Yu Hecheng and the optimized convenience researchers. There are many kinds of MG132 (Z-Leu-Leu-Leu-al, also termed Cbz-LLL or z-LLL) developed by the most widely used inhibitor.1994 (Z-Leu-Leu-Leu-al, also termed Cbz-LLL or z-LLL) is the first kind and a classic proteasome inhibitor. The widely used proteasome biology is widely used in.MG132 is a reversible, effective 26S peptide aldehyde inhibitor. To inhibit the degradation of protein on the proteasome pathway, and then affect cell proliferation and promote cell apoptosis. In natural inhibitors, Lactacystin is a well known, cell permeable proteasome inhibitor. It is a metabolite of Streptomyces in the soil, and is a selective inhibitor of the 20S proteasome. The beta 2 lactone of lactomome and its active intermediate can selectively and irreversibly bind to the beta 5 subunit of the mammalian proteasome and then inhibit the activity of a variety of peptidases. Autophagy lysosome system is involved in removing disease proteins and removing aggregated proteins or damaged cells such as mitochondria. In neurodegenerative disease, it is easy to gather. The disease protein of the collection is usually resistant to the degradation of proteasome and is more inclined to degrade.3MA (3-Methyladenine) through autophagy - lysosome system. It is a widely used autophagy inhibitor, which inhibits autophagy by inhibiting the formation of autophagosomes by inhibiting PI3K. The 3-Methyladenine (Bafilomycin A1) is a source of grey streptomycin The vesicular H+-ATP enzyme inhibitor, a macrolide antibiotic, is a macrolide antibiotic that inhibits vesicle acidification and inhibits the formation of autophagoside. Rapamycin (Rapamycin) is a novel macrolide antibiotic isolated from actinomycetes culture. It inhibits rapamycin by forming a complex of FK506 binding protein (FKBP12). The activation of mammalian target of rapamycin (m TOR) leads to autophagy activation. Previous studies have shown that TDP-43 can be degraded through the ubiquitin proteasome pathway and autophagy lysosome pathway, but the degradation of aggregates in the stable transfected TDP-25 cell lines and its toxicity are still not reported. The first part is stable to TDP-. The establishment and identification of 25 cell model: the purpose of this study is to establish a cell model for stable transfection of TDP-25, and then identify whether it is successful. Methods: the empty P CI-neo plasmid and TDP-25c DNAs plasmid were transiently transfected into NSC34 cells by liposome transfection according to the operation process. After transfection of plasmid to plasmid 48 h, the culture solution was replaced. For culture medium containing G418, 3 weeks later, the polyclonal cells resistant to the expression plasmid of G418 were selected. The transfected plasmids were isolated by flow cytometry, that is, empty plasmid cells with green fluorescent labels and TDP-25 cells. The expression of plasmids in cells was verified by Western blot; the distribution characteristics of TDP-25 protein were observed by inverted fluorescence microscope and laser confocal microscope; the morphology of the aggregates in the stable TDP-25 cells was observed by transmission electron microscope; MDA method was used to detect the lipid peroxidation in the stable cell lines; and TDP-25 cells were detected by CCK-8 technology. The activity of Mito Tracker#174; Red CM-H2XRos detection of reactive oxygen species; WB method to detect the expression of apoptotic markers to react to the toxic effect of stable cell lines. Results: (1) the.Western blot data of the stable transfected TDP-25 cell line and empty plasmid cell line can be obtained by flow cytometry. The expression level of TDP-25 is approximately the same. The 47%. (2) of endogenous TDP-43 in the stable cell line, TDP-25 protein is located both in the nucleus and in the cytoplasm. After the proteasome inhibitor MG132 is given, TDP-25 is more likely to form dense aggregates in the nucleus and cytoplasm. Under transmission electron microscope, the electron dense particles of fibrous bundles are observed by the transmission electron microscope. (3) stable transfection of TDP-25 There are oxidative stress, lipid peroxidation and apoptosis in the cells. (4) by laser confocal microscopy, the aggregation can be Co located with autophagy related protein P62 and ubiquitin in TDP-25 stable cells. Conclusion: the stable transfection cell line is established by flow cytometry. Compared with the control cells, the stable transfection of TDP-25 Cells can cause oxidative stress, lipid peroxidation and apoptosis. Second the effect of part of the ubiquitin proteasome system on TDP-25 cells: the purpose of this study is to explore the effect of ubiquitin proteasome system on the aggregation of TDP-25 cells in stable transfection and whether TDP-25 cells are toxic. The cell line was established by the instrument separation technique, and the cells were passed into the six pore plate. The proteasome inhibitor MG132 or lactoin was given to observe whether the aggregates were degraded through the ubiquitin proteasome pathway. After 3,6,12,24 hours respectively, the changes of the TDP-25 aggregates or the number of GFP were observed by inverted fluorescence microscopy. The expression of TDP-25 protein was measured by Western blot. The condition of mitochondria of the stable cell line was observed by transmission electron microscope. Results: (1) the time dependent expression of TDP-25 protein increased gradually in the stable TDP-25 cells, and reached the peak after the intervention of the proteasome inhibitor for 12 hours. However, there was no change in the endogenous TDP-43 and the empty plasmid control groups. (2) the number of TDP-25 fluorescence in the stable cells was time dependent and the fluorescence of the proteasome inhibitor reached 3000. in 24 hours, but the fluorescence of the empty plasmid group was not significantly changed. (3) when the TDP-25 was over, it had the properties that were easily aggregated. In stable transfection, the transfection of TDP was stable. In the -25 cell line, we found that TDP-25 formed some small spot aggregates near the nuclear membrane. However, after the proteasome inhibitor MG132 stem, the TDP-25 formed a larger aggregation in the cytoplasm and nucleus. (4) the mitochondria in the stable TDP-25 cells were swollen and the mitochondrial crista dilated. After giving proteasome inhibitors, mitochondria This change is more obvious. Conclusion: the degradation of the aggregates in the stable transfected TDP-25 cell line is mainly through the ubiquitin proteasome system and its mitochondria are abnormal. The toxicity of this cell line depends on the activity of proteasome. The purpose of the third part autophagy lysosome system to stabilize the TDP-25 cells is to explore the purpose of this study. To discuss the effect of autophagy - lysosome system on the stable expression of TDP-25 cells. Methods: using the stable cell line established by flow cytometry, the cells with good growth state were passed into the six pore plates, and the autophagic pathway blockers (buffalamycin A1 and 3MA) and the autophagy pathway activator (rapamycin) were selected respectively. To observe whether the TDP-25 aggregates were degraded by autophagic pathway in the stable cells. The changes in the number of aggregates were observed by inverted fluorescence microscopy, and the expression of TDP-25 protein was measured by Western blotting. Results: (1) different concentrations of the autophagy pathway inhibitor, buffalomycin A1, respectively After intervention of 24 h with 3MA, the morphology, location and number of aggregates were not detected by inverted fluorescence microscopy and Western blotting. (2) after the treatment of 24h with different concentration of autophagy activator rapamycin, the morphology and location of the aggregates were still not detected by inverted fluorescence microscopy and Western blotting. Conclusion: the degradation of aggregates in stably transfected TDP-25 cell lines may not be mediated by autophagy lysosome system.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R744.8

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