稳转TDP-25细胞模型的建立以及TDP-25在肌萎缩侧索硬化症中的相关研究
发布时间：2018-07-29 08:07
【摘要】：背景:肌萎缩侧索硬化[Amyotrophic lateral sclerosis,(ALS)],是一种致死性的运动神经元变性疾病,引起渐进性肌肉无力、瘫痪和过早死亡。同时ALS是一种多因素疾病,有病因学异质性和高度可变的临床表现。它的特征是选择性上下运动神经元变性和死亡,中年起病,在1-5年渐进性瘫痪和死亡。最近,在肌萎缩侧索硬化和伴有泛素阳性包涵体的额颞叶痴呆患者[frontotemporal lobar dementia with ubiquitin-positive inclusions,(FTLD-U)]以及帕金森氏病、路易体痴呆及30%的阿尔茨海默氏病患者的脑组织中发现了TARDNA结合蛋白43 k Da(TDP-43)阳性包涵体。TDP-43是一种保守的,广泛表达的核蛋白,由1号染色体上的TARDBP基因编码。在结构上有5个功能区域,包括2个RNA识别模体(RRM1和RRM2),一个富含甘氨酸区域(GRR),一个核定位信号(NLS)和细胞核输出信号(NES)来介导核与胞质的穿梭运动。功能上与外显子跳跃和选择性剪接有关。病理性TDP43从核再分布到细胞质,在胞质聚集。自从2006年在散发的ALS患者和FTLD患者中发现以TDP-43作为主要组分的泛素蛋白聚集体,为我们理解ALS的发病机制补充了一个主要的视野。随后的研究发现,散发性和家族性FTLD-U和ALS患者以胞质内聚集不溶性的、过度磷酸化的、泛素化的和蛋白水解裂开的C端片段在受累的脑和脊髓区域堆积为特征。尤其是25kd的TDP-43 C末端片段(TDP-25)在受累的脑区域聚集提示它可能涉及疾病的发病机制。TDP-43在RNA代谢、神经突起增长、神经元发育以及应激颗粒组成中起重要作用。研究表明TDP-25过度表达足以引起TDP-43的错误定位和胞质内堆积内源性全长TDP-43。但是在ALS相关的细胞模型中,仍未建立稳定转染TDP-25的细胞系。ALS的一个病理特点为:在运动神经元内有异常的聚集体,而且TDP-43包涵体中富含TDP-43的C末端片段。在真核细胞中,关于蛋白质的清除主要有两个系统:泛素蛋白酶体系统和自噬-溶酶体系统。泛素蛋白酶体系统在维持细胞蛋白质平衡上起重要作用,参与多个细胞过程,比如细胞周期、细胞的分化和发展、应激反应和DNA修饰。许多神经变性病在脑组织形成泛素化的包涵体就是通过这个途径来降解的。TDP-43位于脑组织的泛素阳性包涵体内,表明泛素蛋白酶体系统可能参与TDP-43的降解或它的发病机制。由于化学结构的不同,蛋白酶体抑制剂大致可以分为两类,天然产物和合成类似物。肽醛类抑制剂是被确定的第一种蛋白酶体抑制剂,由于合成和优化的方便研究人员已经开发出了很多种,他们是使用最广泛的抑制剂。1994年由Rock研制出的MG132(Z-Leu-Leu-Leu-al,also termed Cbz-LLL or z-LLL)是第一种被确定的而且是经典的蛋白酶体抑制剂,在蛋白酶体生物学上被广泛应用。MG132是一种可逆的、有效的26S肽醛类抑制剂,它可以抑制蛋白酶体通路上蛋白质的降解,进而影响细胞的增殖,促进细胞凋亡。在天然抑制剂中,乳胞素(Lactacystin)是众所周知的、细胞可渗透的蛋白酶体抑制剂。它是存在于土壤中的链霉菌属的代谢物,是一种选择性20S蛋白酶体的抑制剂。乳胞素与其活性中间体的β2内酯可以选择性地、不可逆地结合于哺乳动物的蛋白酶体的β5亚单位,进而抑制多种肽酶的活性。自噬-溶酶体系统参与清除疾病蛋白以及除掉聚集的蛋白质或损伤的细胞器如线粒体。在神经变性病,易于聚集的疾病蛋白质通常对于蛋白酶体的降解有抵抗,更倾向于通过自噬-溶酶体系统降解。3MA(3-Methyladenine),是一种被广泛使用的自噬抑制剂,通过抑制PI3K阻断自噬体的形成来抑制自噬。巴弗洛霉素A1(Bafilomycin A1)是一种来源于灰色链霉素的囊泡性H+-ATP酶质子泵抑制剂,属于大环内酯类抗生素,它可以抑制囊泡酸化从而抑制自噬体后期的形成。雷帕霉素(Rapamycin)是一种分离于放线菌培养液中的新型大环内酯类抗生素。它通过形成FK506结合蛋白(FKBP12)的复合物抑制雷帕霉素靶蛋白(mammalian target of rapamycin,m TOR)的激活,从而导致自噬激活。以往的研究表明TDP-43可以通过泛素-蛋白酶体途径和自噬-溶酶体途径降解,但是对于稳定转染TDP-25细胞系中聚集体的降解以及是否有毒性仍未见报道。第一部分稳转TDP-25细胞模型的建立及鉴定目的:本研究的目的是建立稳定转染TDP-25的细胞模型,然后鉴定是否成功。方法:按照操作流程,应用脂质体转染的方法在NSC34细胞中瞬时转染empty p CI-neo质粒和TDP-25c DNAs质粒。在细胞转染质粒48 h后,将培养液更换为含有G418的培养液培养,3周后挑选抵抗G418的表达质粒的多克隆细胞。用流式细胞仪分选技术分离出转染质粒的细胞,即带有绿色荧光标签的空质粒细胞以及TDP-25细胞。培养分选出的表达TDP-25的多克隆细胞,收集生长状态良好的细胞并用Western印迹方法验证细胞中质粒的表达;用倒置荧光显微镜及激光共聚焦显微镜观察TDP-25蛋白的分布特点;使用透射电子显微镜观察稳转TDP-25细胞中聚集体的形态;MDA方法检测稳转细胞系的脂质过氧化作用;用CCK-8技术检测TDP-25细胞的活力;Mito Tracker#174;Red CM-H2XRos检测活性氧;WB方法检测凋亡标志物的表达来反应稳转细胞系的毒性作用。结果:(1)通过流式细胞仪分选技术可以获得稳定转染的TDP-25细胞系和空质粒细胞系。Western印迹数据显示:TDP-25的表达水平大约是内源性TDP-43的47%。(2)在稳转的细胞系中,TDP-25蛋白既位于细胞核中,也可位于细胞浆中。给予蛋白酶体抑制剂MG132后TDP-25在胞核和胞浆更容易形成致密的聚集体。透射电子显微镜下观察聚集体为纤维束状的电子致密颗粒。(3)稳定转染TDP-25的细胞存在氧化应激、脂质过氧化和细胞凋亡。(4)通过激光共聚焦显微镜观察,在TDP-25稳转细胞中,聚集体可以与自噬相关蛋白P62以及泛素进行共定位。结论:通过流式细胞仪分选技术建立了稳转细胞系。和对照细胞相比,稳定转染TDP-25的细胞可以引起氧化应激、脂质过氧化和凋亡。第二部分泛素-蛋白酶体系统对稳转TDP-25细胞的影响目的:本研究的目的是探讨泛素-蛋白酶体系统对稳定转染TDP-25细胞中聚集体的影响以及TDP-25细胞是否存在毒性。方法:使用通过流式细胞仪分选技术建立的稳转细胞系,将细胞传代至六孔板中,分别给予蛋白酶体抑制剂MG132或乳胞素后观察聚集体是否通过泛素-蛋白酶体通路进行降解。在分别干预3、6、12、24小时后,使用倒置荧光显微镜观察TDP-25聚集体或GFP荧光数量的变化,并收集细胞使用Western印迹方法对TDP-25蛋白质的表达进行定量测定。使用透射电子显微镜观察稳转细胞系的线粒体的情况。结果:(1)在稳转TDP-25细胞中,TDP-25蛋白质的表达呈时间依赖性逐渐增加,在蛋白酶体抑制剂干预12小时后达到高峰。然而,内源性TDP-43和空质粒对照组没有发现改变。(2)在稳转细胞中TDP-25的荧光数量呈时间依赖性增加,在蛋白酶体抑制剂干预24小时达到3000。然而空质粒组的荧光没有明显改变。(3)当TDP-25过表达时,它具有易于聚集的性质。在稳定转染TDP-25细胞系,我们发现TDP-25在核膜附近形成一些小的点状的聚集体。然而,经蛋白酶体抑制剂MG132干预后,TDP-25在细胞质和细胞核形成更大的聚集体。(4)稳转TDP-25细胞中的线粒体是肿胀的,线粒体嵴是扩张的。给予蛋白酶体抑制剂后,线粒体的这种改变更加明显。结论:稳定转染TDP-25细胞系中聚集体的降解主要是通过泛素-蛋白酶体系统,其线粒体是异常的。这个细胞系的毒性依赖于蛋白酶体的活性。第三部分自噬-溶酶体系统对稳转TDP-25细胞的影响目的:本研究的目的是探讨自噬-溶酶体系统对稳定表达TDP-25细胞的影响。方法:使用通过流式细胞仪分选技术建立的稳转细胞系,将生长状态良好的细胞传代至六孔板中,分别选用不同浓度的自噬通路阻断剂(巴弗洛霉素A1和3MA)和自噬通路激活剂(雷帕霉素)处理细胞,观察稳转细胞中TDP-25聚集体是否通过自噬通路降解。使用倒置荧光显微镜观察聚集体荧光数量的变化,并收集细胞使用Western印迹方法对TDP-25蛋白的表达进行定量测定。结果:(1)分别使用不同浓度的自噬途径抑制剂巴弗洛霉素A1和3MA干预稳转细胞24 h后,倒置荧光显微镜及Western印迹方法均未检测到聚集体形态、位置及数量的改变。(2)分别使用不同浓度的自噬途径激活剂雷帕霉素处理稳转细胞24h后,倒置荧光显微镜及Western印迹方法仍未检测到聚集体形态、位置及数量的改变。结论:稳定转染TDP-25细胞系中聚集体的降解可能不通过自噬-溶酶体系统。
[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.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R744.8
[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.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R744.8
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