在蛋白水平和mRNA水平检测线粒体分裂蛋白Drp1在小鼠中枢神经系统的分布特点

发布时间：2018-06-04 12:52

本文选题：Drp1蛋白 + Drp1　；参考：《成都医学院》2017年硕士论文

【摘要】：具有双层膜结构的细胞器——线粒体(Mitochondria)是维持生命活动的关键结构,为细胞持续供能,决定着细胞的命运。神经元作为高度极化细胞,是高能量需求结构,研究证明,线粒体通过提供能量、维持钙平衡、产生活性氧和诱导凋亡等,在神经元的发育及功能中发挥重要作用。其中,线粒体数量、质量和分布所组成的线粒体动力学是其重要的结构基础,而线粒体融合与分裂的异常被证明参与了多种神经退行性疾病的发生与发展过程。与多个GTPase蛋白家族成员参与线粒体融合机制不同的是,目前仅证明一种GTPase蛋白家族成员,即动力相关蛋白1(dynamin-related protein 1,Drp1),负责执行神经系统内细胞的线粒体分裂。然而,对于Drp1在神经系统不同区域的分布情况、在亚细胞器的定位情况以及在神经元不同结构中的分布情况,目前均不清楚,缺乏系统研究,阻碍了对生理状态下神经系统线粒体动力学变化的清楚认识,影响了对线粒体功能异常参与多种神经病理性改变的神经和分子机制的深入理解。目的本研究通过研究小鼠中枢神经系统(脑和脊髓)内Drp1分子分别在蛋白水平和mRNA水平的分布情况,试图回答以下问题:第一,中枢神经系统Drp1分布是广泛还是局限,是否存在区域特异性?第二,Drp1在神经元和胶质细胞是否分布,是否存在细胞特异性?第三,Drp1在胞核、胞体、线粒体、内质网、核糖体是否分布,是否存在亚细胞器定位特异性?第四,Drp1在神经元胞体、树突、轴突以及突触是否分布,是否存在结构特异性?材料与方法利用成年雄性野生型C57BL/6小鼠和谷氨酸脱羧酶67-绿色荧光蛋白(glutamic acid decarboxylase 67-green fluorescent protein,GAD67-GFP)基因敲入小鼠,综合采用了线粒体示踪剂Mito-Red鞘内注射、免疫印记(Western blot,WB)、免疫荧光多重染色(immunofluorescence,IF)、免疫荧光原位杂交(fluorescence in situ hybridization,FISH)、免疫电镜(immunoelectron microscopy,IEM)等技术。结果drp1的蛋白水平和mrna水平均在小鼠中枢神经系统广泛分布,drp1蛋白与drp1mrna分布趋势基本一致,但就表达数量及特异性来说,drp1蛋白不如drp1mrna。利用drp1蛋白的单克隆抗体以及神经元标志物neun抗体进行免疫荧光双重染色,观察到drp1荧光阳性细胞多表达neun,结合已有研究证明,drp1是神经元特异性的蛋白。drp1的蛋白分布具有区域特异性,drp1表达量最高的部位包括:隔核、苍白球、外侧膝状体、丘脑、丘脑网状结构、脑干网状结构、黑质、腹侧被盖、中缝核群、蓝斑、barrington核、耳蜗核、面神经核、三叉神经脊核、外侧楔核。而在皮质i-ii层、新纹状体区、海马等表达最弱,在小脑,drp1专一表达于浦肯野细胞层。另外,westernblot实验也说明drp1的蛋白分布具有区域特异性。鉴于drp1蛋白和mrna强阳性部位中的小脑浦肯野细胞层、三叉神经脊核(spvi)、小细胞网状核(parn)、孤束旁核(pas)、丘脑网状核(rt)和面运动神经核团(vii)等区域既往被证明是脑内抑制性神经元gaba能神经元聚集的区域,本研究利用gad67-gfpknock-in小鼠检测了drp1在以上区域gfp阳性细胞上的表达情况。结果表明,drp1可能分布于大部分gaba能神经元,对脑内的抑制性神经元的功能起到重要的调节作用。本研究利用mito-red鞘内注射结合免疫荧光染色方法,观察了drp1的亚细胞结构的分布情况。mito-red阳性结构全部与drp1阳性结构形成共存,但是有大量drp1结构上未见红色荧光。以上结果证实,drp1的表达主要位于非线粒体的神经元胞浆内。利用免疫电镜方法,在电镜下观察了drp1在海马、小脑以及pag的亚细胞结构的分布情况。结果表明drp1阳性胶体金颗粒在单个神经元中均较少,呈散在分布。金颗粒主要位于胞浆中线粒体之间的结构,有的位于内质网,有的位于核糖体,但是很少位于线粒体,在细胞膜上几乎均未见阳性颗粒,这与光镜下的结果相一致。以上结果表明,drp1的分布具有一定的亚细胞结构特异性。结论Drp1通过调控线粒体分裂参与了机体的多种功能,包括机体正常睡眠觉醒规律的维持、学习记忆的保障、内脏活动的调节、听觉信息的加工以及对痛等感觉的觉知及加工等,可见Drp1对机体正常功能的维持有着至关重要的作用。但是Drp1调控的具体机制尚需进一步研究。研究表明,Drp1蛋白在生理状态下多位于胞浆,仅有3%左右位于线粒体,提示介导Drp1募集到线粒体启动线粒体分裂的分子基础可能是将来研究的重要内容。本研究还发现,Drp1主要分布于神经元的胞体和树突结构,而鲜见于轴突,提示聚集有大量线粒体的轴突结构可能需要其他分子进行线粒体动力学调控。但是,有关Drp1在不同状态下的功能及其调控尚需进一步研究。
[Abstract]:The mitochondria (Mitochondria), the key structure of the double membrane structure, is the key structure for the maintenance of life activities. It determines the fate of the cells for the continuous energy supply of cells. As a highly polarized cell, the neuron is a high energy demand structure. The research has proved that mitochondria can maintain calcium balance, produce reactive oxygen species and induce apoptosis by providing energy. The mitochondrial dynamics, composed of mitochondria, mass and distribution, is an important structural basis for the development and function of neurons. The abnormalities of mitochondrial fusion and division have been proved to be involved in the occurrence and development of a variety of neurodegenerative diseases. Multiple GTPase protein family members participate in mitochondria. The fusion mechanism is different from the fact that only a member of the GTPase protein family, the dynamin-related protein 1 (Drp1), is responsible for the mitochondrial division of the cells in the nervous system. However, the distribution of Drp1 in different regions of the nervous system, the location of the subcellular organelles and the different structures of the neurons in the neurons. The distribution of the medium is not clear at present. Lack of systematic study hinders a clear understanding of the changes in the mitochondrial dynamics of the nervous system in physiological state, and affects the deep understanding of the neural and molecular mechanisms involved in the abnormal involvement of mitochondria in various neuropathic changes. The distribution of Drp1 molecules in the protein level and mRNA level in the spinal cord, respectively, tries to answer the following questions: first, whether the distribution of Drp1 in the central nervous system is wide or limited, is there a regional specificity? Second, whether Drp1 is distributed in neurons and glia, is there a cell specificity? Third, Drp1 in the nucleus, the cell body, and the mitochondria Whether or not the endoplasmic reticulum and ribosomes are distributed, is there a specific specificity of the subcellular localization? Fourth, whether Drp1 is distributed in the cell body, dendrites, axons and synapses, is there a structural specificity? Materials and methods use adult male wild type C57BL/6 mice and glutamic acid decarboxylase 67-g (glutamic acid decarboxylase 67-g). Reen fluorescent protein, GAD67-GFP) gene knocked into mice, combined with mitochondrial tracer Mito-Red intrathecal injection, immune imprint (Western blot, WB), immunofluorescence multiplex staining (immunofluorescence, IF), immunofluorescent in situ hybridization (fluorescence in), immune electron microscopy, etc. Results the protein level and mRNA level of drp1 were widely distributed in the central nervous system of mice. The distribution trend of drp1 protein and drp1mrna was basically the same, but in terms of the number and specificity of the expression, the drp1 protein was not as good as the monoclonal antibody of the drp1mrna. using the drp1 protein and the immunofluorescence double staining of the NeuN antibody of the neuron. It was found that drp1 fluorescent positive cells expressed more NeuN. Combining with previous studies, drp1 is a neuron specific protein.Drp1 protein distribution with regional specificity. The highest levels of drp1 expression include: septum, globus pallidus, lateral geniculate body, thalamus, thalamus reticular structure, brain stem reticular structure, substantia nigra, ventral tegmentum, raphe nucleus, locus blue-spot, bar The rington nucleus, the cochlear nucleus, the nucleus of the facial nerve, the trigeminal nucleus, and the lateral cuneate are the weakest in the cortex I-II, the new striatum and the hippocampus, and in the cerebellum, the drp1 is expressed in the pukini cell layer. In addition, the Westernblot experiment also shows that the protein distribution of drp1 is regional specific. In view of the cerebellum of the strong positive parts of the drp1 and mRNA, the cerebellum The Ken field cell layer, the trigeminal nucleus (spvi), the small cell reticular nucleus (PARN), the paramnicular nucleus (PAS), the thalamus reticular nucleus (RT) and the facial motor nucleus group (VII) have previously been proved to be the areas of the aggregation of the GABA energy neurons in the cerebral inhibitory neurons. This study used gad67-gfpknock-in mice to detect the GFP positive cells of drp1 in the above region. The results show that drp1 may be distributed in most of the GABA energy neurons and plays an important role in the function of the inhibitory neurons in the brain. The distribution of subcellular structures of drp1 is observed by mito-red intrathecal injection combined with immunofluorescence staining, and all of the.Mito-red positive structures of the drp1 structure are all with the positive structure of the positive structure of the.Mito-red. The results showed that the expression of drp1 was mainly located in the cytoplasm of non mitochondrial neurons. The distribution of the subcellular structure of drp1 in the hippocampus, cerebellum and PAG was observed under electron microscopy. The results showed that the drp1 positive colloid gold particles were in a single nerve under the electron microscope. The gold particles are mainly located in the structure of mitochondria in the cytoplasm, some are in the endoplasmic reticulum, some are located in ribosomes, but few are in the mitochondria, and almost no positive particles are found on the membrane. This is in accordance with the results under light microscope. The above results show that the distribution of drp1 has a certain subcellular structure. Conclusion Drp1 participates in various functions of the body by regulating mitochondrial division, including the maintenance of normal sleep awareness, the protection of learning and memory, the regulation of visceral activity, the processing of the auditory information and the perception and processing of pain and so on. It can be seen that Drp1 has a vital role in the maintenance of normal function of the body. But the specific mechanism of Drp1 regulation still needs further study. The study shows that Drp1 protein is mostly located in the cytoplasm and only about 3% in the mitochondria. It suggests that the molecular basis of Drp1 recruitment to mitochondria to initiate mitochondrial division may be an important part of future research. This study also found that Drp1 is mainly distributed in neurons. The structure of the cell body and dendrite, which is rarely seen in the axon, suggests that the axon structure of a large number of mitochondria may require other molecules to regulate the mitochondrial dynamics. However, the function and regulation of Drp1 in different states still need to be further studied.
【学位授予单位】：成都医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R338

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