本文選題：Drp1蛋白 + Drp1��； 參考：《成都醫(yī)學(xué)院》2017年碩士論文

【摘要】：具有雙層膜結(jié)構(gòu)的細(xì)胞器——線粒體(Mitochondria)是維持生命活動(dòng)的關(guān)鍵結(jié)構(gòu),為細(xì)胞持續(xù)供能,決定著細(xì)胞的命運(yùn)。神經(jīng)元作為高度極化細(xì)胞,是高能量需求結(jié)構(gòu),研究證明,線粒體通過提供能量、維持鈣平衡、產(chǎn)生活性氧和誘導(dǎo)凋亡等,在神經(jīng)元的發(fā)育及功能中發(fā)揮重要作用。其中,線粒體數(shù)量、質(zhì)量和分布所組成的線粒體動(dòng)力學(xué)是其重要的結(jié)構(gòu)基礎(chǔ),而線粒體融合與分裂的異常被證明參與了多種神經(jīng)退行性疾病的發(fā)生與發(fā)展過程。與多個(gè)GTPase蛋白家族成員參與線粒體融合機(jī)制不同的是,目前僅證明一種GTPase蛋白家族成員,即動(dòng)力相關(guān)蛋白1(dynamin-related protein 1,Drp1),負(fù)責(zé)執(zhí)行神經(jīng)系統(tǒng)內(nèi)細(xì)胞的線粒體分裂。然而,對(duì)于Drp1在神經(jīng)系統(tǒng)不同區(qū)域的分布情況、在亞細(xì)胞器的定位情況以及在神經(jīng)元不同結(jié)構(gòu)中的分布情況,目前均不清楚,缺乏系統(tǒng)研究,阻礙了對(duì)生理狀態(tài)下神經(jīng)系統(tǒng)線粒體動(dòng)力學(xué)變化的清楚認(rèn)識(shí),影響了對(duì)線粒體功能異常參與多種神經(jīng)病理性改變的神經(jīng)和分子機(jī)制的深入理解。目的本研究通過研究小鼠中樞神經(jīng)系統(tǒng)(腦和脊髓)內(nèi)Drp1分子分別在蛋白水平和mRNA水平的分布情況,試圖回答以下問題:第一,中樞神經(jīng)系統(tǒng)Drp1分布是廣泛還是局限,是否存在區(qū)域特異性?第二,Drp1在神經(jīng)元和膠質(zhì)細(xì)胞是否分布,是否存在細(xì)胞特異性?第三,Drp1在胞核、胞體、線粒體、內(nèi)質(zhì)網(wǎng)、核糖體是否分布,是否存在亞細(xì)胞器定位特異性?第四,Drp1在神經(jīng)元胞體、樹突、軸突以及突觸是否分布,是否存在結(jié)構(gòu)特異性?材料與方法利用成年雄性野生型C57BL/6小鼠和谷氨酸脫羧酶67-綠色熒光蛋白(glutamic acid decarboxylase 67-green fluorescent protein,GAD67-GFP)基因敲入小鼠,綜合采用了線粒體示蹤劑Mito-Red鞘內(nèi)注射、免疫印記(Western blot,WB)、免疫熒光多重染色(immunofluorescence,IF)、免疫熒光原位雜交(fluorescence in situ hybridization,FISH)、免疫電鏡(immunoelectron microscopy,IEM)等技術(shù)。結(jié)果drp1的蛋白水平和mrna水平均在小鼠中樞神經(jīng)系統(tǒng)廣泛分布,drp1蛋白與drp1mrna分布趨勢(shì)基本一致,但就表達(dá)數(shù)量及特異性來說,drp1蛋白不如drp1mrna。利用drp1蛋白的單克隆抗體以及神經(jīng)元標(biāo)志物neun抗體進(jìn)行免疫熒光雙重染色,觀察到drp1熒光陽性細(xì)胞多表達(dá)neun,結(jié)合已有研究證明,drp1是神經(jīng)元特異性的蛋白。drp1的蛋白分布具有區(qū)域特異性,drp1表達(dá)量最高的部位包括:隔核、蒼白球、外側(cè)膝狀體、丘腦、丘腦網(wǎng)狀結(jié)構(gòu)、腦干網(wǎng)狀結(jié)構(gòu)、黑質(zhì)、腹側(cè)被蓋、中縫核群、藍(lán)斑、barrington核、耳蝸核、面神經(jīng)核、三叉神經(jīng)脊核、外側(cè)楔核。而在皮質(zhì)i-ii層、新紋狀體區(qū)、海馬等表達(dá)最弱,在小腦,drp1專一表達(dá)于浦肯野細(xì)胞層。另外,westernblot實(shí)驗(yàn)也說明drp1的蛋白分布具有區(qū)域特異性。鑒于drp1蛋白和mrna強(qiáng)陽性部位中的小腦浦肯野細(xì)胞層、三叉神經(jīng)脊核(spvi)、小細(xì)胞網(wǎng)狀核(parn)、孤束旁核(pas)、丘腦網(wǎng)狀核(rt)和面運(yùn)動(dòng)神經(jīng)核團(tuán)(vii)等區(qū)域既往被證明是腦內(nèi)抑制性神經(jīng)元gaba能神經(jīng)元聚集的區(qū)域,本研究利用gad67-gfpknock-in小鼠檢測(cè)了drp1在以上區(qū)域gfp陽性細(xì)胞上的表達(dá)情況。結(jié)果表明,drp1可能分布于大部分gaba能神經(jīng)元,對(duì)腦內(nèi)的抑制性神經(jīng)元的功能起到重要的調(diào)節(jié)作用。本研究利用mito-red鞘內(nèi)注射結(jié)合免疫熒光染色方法,觀察了drp1的亞細(xì)胞結(jié)構(gòu)的分布情況。mito-red陽性結(jié)構(gòu)全部與drp1陽性結(jié)構(gòu)形成共存,但是有大量drp1結(jié)構(gòu)上未見紅色熒光。以上結(jié)果證實(shí),drp1的表達(dá)主要位于非線粒體的神經(jīng)元胞漿內(nèi)。利用免疫電鏡方法,在電鏡下觀察了drp1在海馬、小腦以及pag的亞細(xì)胞結(jié)構(gòu)的分布情況。結(jié)果表明drp1陽性膠體金顆粒在單個(gè)神經(jīng)元中均較少,呈散在分布。金顆粒主要位于胞漿中線粒體之間的結(jié)構(gòu),有的位于內(nèi)質(zhì)網(wǎng),有的位于核糖體,但是很少位于線粒體,在細(xì)胞膜上幾乎均未見陽性顆粒,這與光鏡下的結(jié)果相一致。以上結(jié)果表明,drp1的分布具有一定的亞細(xì)胞結(jié)構(gòu)特異性。結(jié)論Drp1通過調(diào)控線粒體分裂參與了機(jī)體的多種功能,包括機(jī)體正常睡眠覺醒規(guī)律的維持、學(xué)習(xí)記憶的保障、內(nèi)臟活動(dòng)的調(diào)節(jié)、聽覺信息的加工以及對(duì)痛等感覺的覺知及加工等,可見Drp1對(duì)機(jī)體正常功能的維持有著至關(guān)重要的作用。但是Drp1調(diào)控的具體機(jī)制尚需進(jìn)一步研究。研究表明,Drp1蛋白在生理狀態(tài)下多位于胞漿,僅有3%左右位于線粒體,提示介導(dǎo)Drp1募集到線粒體啟動(dòng)線粒體分裂的分子基礎(chǔ)可能是將來研究的重要內(nèi)容。本研究還發(fā)現(xiàn),Drp1主要分布于神經(jīng)元的胞體和樹突結(jié)構(gòu),而鮮見于軸突,提示聚集有大量線粒體的軸突結(jié)構(gòu)可能需要其他分子進(jìn)行線粒體動(dòng)力學(xué)調(diào)控。但是,有關(guān)Drp1在不同狀態(tài)下的功能及其調(diào)控尚需進(jìn)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.
【學(xué)位授予單位】：成都醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R338

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