线粒体MEF2D氧化修饰水平变化在PD发病中的作用

发布时间：2017-12-31 11:34

本文关键词：线粒体MEF2D氧化修饰水平变化在PD发病中的作用　出处：《第四军医大学》2015年硕士论文　论文类型：学位论文

【摘要】：帕金森病(PD)是一种神经退行性疾病,其主要临床特征是严重影响患者的运动功能。随着我国逐渐进入老龄化时代,PD越来越受到家庭与社会的关注[1,2]。PD的主要病理学特点是中脑SNc区DA神经元发生变性和死亡,导致多巴胺的释放减少,但DA神经元变性死亡的具体机制尚不明确。研究发现,约5%-10%的PD例明确是和遗传因素相关的,这部分病例与特定的基因改变有关[3],而大多数PD病例是散发的,其确切的发病原因还不明确。流行病学研究已经证明特定的环境因素暴露(如神经毒剂)和PD的发病之间有确定的联系[4],表明部分神经毒剂会导致或促进PD发病。现在有一个被广泛认同的观点是:遗传与环境因素共同作用于细胞,破坏细胞稳态并导致了氧化应激,继而造成了DA神经元的死亡,这其中一个关键的靶点是线粒体。越来越多的实验数据表明,家族遗传性PD中一些基因的突变可能是通过影响线粒体功能而产生致病效果,同样,一些毒剂也是通过这个途径导致PD发病。但是目前对线粒体功能紊乱、氧化应激级联反应、蛋白聚集和诱发神经元死亡的关键分子及信号通路缺乏进一步的了解,妨碍了PD的诊断治疗,特别是早期发现疾病。神经影像学、基因组学和系统生物学的方法在反映疾病的发展和变化上仍存在较多的限制和障碍[5]。在发病机制深入研究基础上,探索一种可靠、敏感的PD生物学标记物成为目前急需解决的关键问题。肌细胞增强因子2(MEF2)最早在骨骼肌的相关研究中被发现,后来被证实在越来越多的方面发挥自己特有的作用。MEF2D已被证实与神经元存活密切相关[6,7]。本课题组前期研究发现,转录因子MEF2D对于DA神经元的存活起关键性作用[6],不同的细胞应激能够通过相应的信号通路调节MEF2D的功能,对神经元的存活产生影响。MEF2D在DA神经元线粒体中表达,并参与呼吸链复合体I活性的调节[7]。在线粒体中,MEF2D特异性调节线粒体NADH脱氢酶6(ND6)基因的转录表达。ND6是唯一由线粒体轻链编码的蛋白质,在呼吸链复合体I组装过程中发挥着必不可少的作用;降低线粒体MEF2D表达水平会明显抑制呼吸链复合体I的功能,导致细胞内过氧化物水平升高,诱发神经元死亡[8],而大量表达线粒体靶向MEF2D时可以保护神经毒素诱发的DA神经元死亡。现有研究结果提示:线粒体MEF2D对与线粒体功能具有重要调控作用,MEF2D与其氧化修饰水平对于把金森并的发病过程具有重要影响。本研究采用小鼠MPTP-PD亚急性模型,探索在PD模型中,DA神经元线粒体MEF2D表达水平以及氧化修饰水平的动态变化;研究MEF2D、ND6以及线粒体功能变化与DA神经元损伤的关系;通过与其他转录因子对比,初步分析线粒体MEF2D及其氧化修饰水平作为PD生物学标记物的可行性。实验一:MPTP-PD模型中动态检测线粒体MEF2D水平变化方法(1)建立小鼠MPTP-PD亚急性模型,实验动物随机分为4组:Con(对照)组,D1(给药第1天)组,D3(给药第3天)组,D5(给药第5天)组,D1、D3和D5组为实验组。实验组小鼠按30mg/kg剂量腹腔注射MPTP,注射时间为上午9点,每天一次,连续5天,对照组于同时间注射同等剂量0.9%生理盐水。(2)在给药的当天下午3点处死D1组小鼠,第3天同时间点处死D3组小鼠,第5天同时间点处死Con组和D5组小鼠,分别收取样本。(3)使用免疫荧光染色法动态检测小鼠中脑DA神经元水平变化情况。(4)分离细胞质、细胞核以及线粒体,使用免疫印迹法检测分离纯化效果。(5)使用免疫印迹法动态检测小鼠中脑DA神经元细胞核和线粒体MEF2D水平变化情况。(6)分离小鼠的小脑,皮层,纹状体,使用免疫印迹法分别检测各脑区线粒体MEF2D水平变化情况。结果(1)免疫荧光结果显示,在给药的第1、3、5天中,与对照组相比,小鼠黑质致密部DA神经元水平出现不同程度显著下降,在给药第5天几乎消失殆尽,证明小鼠MPTP-PD亚急性模型建立成功。(2)免疫印迹结果显示细胞质,细胞核以及线粒体分离效果良好,相互之间没有混杂或污染,达到实验要求。(3)在给药的1、3、5天中,细胞核MEF2D水平没有出现明显变化,而同区域的线粒体MEF2D在给药的第一天即出现明显下降(P0.05),并在第3天和第5天出现显著下降(P0.01)。(4)小鼠小脑,皮层,纹状体线粒体MEF2D水平在给药的1、3、5天中没有出现明显变化,只有黑质区线粒体MEF2D水平与对照组相比明显降低(P0.05)。结论(1)在PD模型中,小鼠黑质区线粒体MEF2D水平变化与同时间点中脑DA神经元损失水平相一致,能够较好地反映DA神经元水平变化情况。(2)小鼠黑质区线粒体MEF2D水平变化并不是由非特异性应激反应所引起,其变化水平与DA神经元受损程度密切相关。实验二:MPTP-PD模型中动态观测核转录因子Nurr1和线粒体转录因子TFAM的水平变化方法(1)同实验一建立小鼠MPTP-PD亚急性模型,随机分为4组:Con组,D1组,D3组,D5组。(2)使用免疫印迹法检测小鼠中脑DA神经元Nurr1和TFAM动态水平变化。结果(1)在PD模型中,小鼠中脑多巴胺能神经元细胞核转录因子Nurr1在给药的第1、3、5天中没有出现明显的变化,同区域线粒体转录因子TFAM在给药的第1天和第3天没有明显变化,第5天时出现明显下降(P0.05)。结论(1)在PD模型中,细胞核转录因子Nurr1和线粒体转录因子TFAM这两种重要的存活相关转录因子对于多巴胺能神经元的水平变化都不够敏感,线粒体MEF2D相比于Nurr1和TFAM,对于多巴胺能神经元的损伤更加敏感,能够更好地反映多巴胺能神经元的受损程度。实验三:观察线粒体MEF2D氧化修饰水平与DA神经元线粒体功能变化的相关性方法(1)同实验一建立小鼠MPTP-PD亚急性模型,随机分为4组:Con组,D1组,D3组,D5组。(2)使用Oxy Blot技术检测线粒体MEF2D氧化修饰水平动态变化。(3)使用q RT-PCR技术检测ND6 m RNA水平的动态变化。(4)通过测定在450nm下NADH的氧化速率检测呼吸链复合体I活性的动态变化。结果(1)在PD模型中,与对照组相比,中脑DA神经元线粒体MEF2D氧化修饰水平自给药第3天开始出现明显升高,在给药的1、3、5天中呈逐渐升高趋势。(2)同区域ND6 m RNA水平在给药第3天明显下降(P0.05),在第5天显著下降(P0.01)。(3)同区域Complex I活性在第3天和第5天时出现显著下降(P0.01)。结论(1)在PD模型中,小鼠中脑多巴胺能神经元线粒体MEF2D氧化修饰水平与对照组相比明显升高,同时伴有ND6转录水平的明显下降以及呼吸链复合体I活性显著降低,明确了线粒体MEF2D氧化修饰水平与ND6转录水平以及线粒体功能变化的一致性。
[Abstract]:Parkinson's disease (PD) is a neurodegenerative disease, the main clinical features are seriously affecting the patient's motor function. As China has gradually entered the aging era, PD has received more and more attention to family and society the main pathological features of [1,2].PD is SNc DA 'midbrain neurons degeneration and death, leading to dopamine the release of reduced, but the specific mechanism of DA neuron degeneration and death is not clear. The study found that PD were about 5%-10% and is clearly related to genetic factors, this part of the cases with specific genes related to changes in [3], and most PD cases are scattered hair, its exact pathogenesis is not clear. Epidemiological studies have demonstrated exposure to specific environmental factors (such as nerve agents) there is a definite link between the incidence of [4] and PD, showed that some nerve agents can cause or promote the pathogenesis of PD. There is now a widely accepted view is:. Transfer and environmental factors contribute to the destruction of cells, cellular homeostasis and leads to oxidative stress, resulting in the death of DA neurons, which is one of the key targets of mitochondrial. More and more experimental data showed that the same mutation of some genes in familial PD may be caused by pathogenic effect, effect of mitochondria the function, some agents also through the pathway leading to the pathogenesis of PD. But the mitochondrial dysfunction, oxidative stress cascade, the lack of a better understanding of the key molecular protein aggregation and induced neuronal death and signaling pathways, interferes with the diagnosis and treatment of PD, especially the early detection of the disease. Neuroimaging, genomics and method systems biology there are still many limitations and obstacles [5]. based on in-depth study of the pathogenesis of the disease in reflecting disease development and change, to explore a reliable, sensitive PD biological markers sense becomes the key problem solved urgently. Myocyte enhancer factor 2 (MEF2) was originally discovered in the study of skeletal muscle, later proved to play a role in their own unique.MEF2D have been linked to neuronal survival of [6,7]. is closely related to our previous study found that in more and more. The transcription factor MEF2D in DA neurons play a key role in [6], different cell stress can regulate MEF2D signaling through the corresponding function, which affect the expression of.MEF2D in DA neurons in mitochondria on neuronal survival, and participate in the respiratory chain complex I regulating the activity of [7]. in mitochondria, MEF2D specific regulation of mitochondrial NADH dehydrogenase 6 (ND6) gene expression by mitochondrial.ND6 is the only light chain encoding protein in respiratory chain complex I assembly process plays an indispensable for Use; mitochondria MEF2D expression can inhibit the function of respiratory chain complex I, resulting in increased superoxide levels in cells, [8] induced neuronal death, and a large number of expression can protect the mitochondrial targeting to MEF2D neurotoxin induced DA neuronal death. Existing research results suggest that MEF2D plays an important regulatory role of mitochondria and mitochondrial function, the level of MEF2D and for the oxidative modification plays an important role in the pathogenesis of Higginson. This study used mice in subacute MPTP-PD model exploration in PD model, the DA Neuron Mitochondrial MEF2D expression level and dynamic change of the level of oxidative modification; on MEF2D, ND6 and the relationship between mitochondrial function and change of DA neurons with other transcription factors; the level of contrast, preliminary analysis of mitochondrial MEF2D and its feasibility as the oxidative modification of PD biomarkers. Experiment one: MP Change method in TP-PD model of dynamic detection of the level of mitochondrial MEF2D (1) mice in subacute MPTP-PD model, the experimental animal were randomly divided into 4 groups: Con (control group), D1 group (administered first days), D3 (administered third days) group, D5 (administered fifth days) group, D1, D3 and the D5 group for the experimental group. The experimental group of mice by intraperitoneal injection of 30mg/kg MPTP, injection time is at 9 in the morning, once a day for 5 consecutive days, the control group at the same time injected with the same dose of saline. 0.9% (2) in the administration of 3 PM the same day were D1 mice, third days time the mice of D3 group were killed, and fifth day time after Con group and D5 group respectively for samples. (3) using immunofluorescence dynamic changes of murine mesencephalic DA neurons detected. (4) the separation of cytoplasm, nucleus and mitochondria, the effect of pure separation using Western blot test. (5) the use of immune Western blot detection of small dynamic The changes of rat DA neurons of midbrain nuclei and mitochondria. The level of MEF2D (6) isolated from mouse cerebellum, cortex, striatum, respectively to detect the changes of the level of mitochondrial MEF2D in various brain regions by Western blotting. Results (1) immunofluorescence results showed that, in the administration of the first 1,3,5 days, compared with the control group, the mice black nigra DA neurons appeared different degree of level decreased significantly in the administration of fifth days almost disappeared, that mice in subacute MPTP-PD model was established successfully. (2) the results of Western blot showed that the cytoplasm, nucleus and mitochondria isolation effect is good, no pollution or mixed with each other, meet the requirement of experiment. (3) in drug delivery 1,3,5 day, nuclear MEF2D levels did not show significant changes, and mitochondrial MEF2D in same region on the first day of administration is decreased significantly (P0.05), and in third days and fifth days was significantly decreased (P0.01) (4). Mouse cerebellar cortex, striatum, mitochondrial MEF2D levels did not show significant changes in the administration of 1,3,5 days, only the level of mitochondrial MEF2D in the substantia nigra decreased significantly compared with the control group (P0.05). Conclusion (1) in the PD model, the changes of mitochondrial MEF2D levels in mouse substantia nigra of midbrain DA neurons and at the same time point loss level consistent, can better reflect the changes of DA level. (2) neurons in substantia nigra of mice mitochondrial MEF2D level changes and not caused by nonspecific stress responses, it is closely related to the changes of neurons and DA damage. Experiment two: change the level of dynamic observation of nuclear transcription factor Nurr1 and mitochondrial transcription factor TFAM method the MPTP-PD model (1) the mice in subacute MPTP-PD model, were randomly divided into 4 groups: Con group, D1 group, D3 group, D5 group. (2) the use of Western blot detection of mouse mesencephalic DA neurons Nurr1 and TFAM The dynamic level changes. Results (1) in the PD model, mouse midbrain dopaminergic neurons nuclear transcription factor Nurr1 did not show significant changes in the administration of the first 1,3,5 days in the same area, mitochondrial transcription factor TFAM in the administration of first days and third days did not change significantly, decreased significantly at day fifth (P0.05) conclusion. (1) in the PD model, the nuclear transcription factor Nurr1 and mitochondrial transcription factor TFAM of the two important transcription factors related to the survival of dopaminergic neurons changes are not sensitive, mitochondrial MEF2D compared to Nurr1 and TFAM, is more sensitive to the damage of dopaminergic neurons can better reflect dopaminergic neurons the extent of the damage. Experiment three: method of correlation between observed changes of mitochondrial oxidative modification of MEF2D and DA neurons of mitochondrial function (1) with the established mouse MPTP-PD model with subacute. The machine is divided into 4 groups: Con group, D1 group, D3 group, D5 group. (2) detection of mitochondrial MEF2D oxidative modification changes using Oxy Blot technology. (3) using the Q RT-PCR technique to detect the dynamic changes of ND6 m RNA. (4) the dynamic determination of oxidation rate of detection of respiratory chain complex NADH under the 450nm I activity. Results (1) in PD model, compared with the control group, DA neurons of midbrain mitochondrial MEF2D oxidative modification level supply drug appeared third days increased significantly, in the administration of 1,3,5 days increased gradually. (2) with ND6 m RNA in the level of regional administration third the day was significantly decreased (P0.05), decreased significantly after fifth days (P0.01). (3) with the regional Complex I activity in third days and fifth days were significantly reduced (P0.01). Conclusion (1) in the PD model, mouse midbrain dopaminergic neurons oxidative modification of mitochondrial MEF2D levels compared with the control group increased significantly. At the same time with ND6 The transcriptional level decreased significantly, and the activity of I in respiratory chain complex decreased significantly. The consistency of mitochondrial MEF2D oxidative modification level with ND6 transcriptional level and mitochondrial function was confirmed.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R742.5

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