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研究生:黃元駿
研究生(外文):Yuan-Chun Huang
論文名稱:探討NRIP在運動神經元的角色
論文名稱(外文):The role of NRIP in motor meuron.
指導教授:陳小梨陳小梨引用關係
口試委員:王培育符文美李立仁
口試日期:2013-07-31
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:英文
論文頁數:52
中文關鍵詞:核受體交互作用蛋白運動神經元神經膠質化乙醯膽鹼轉移酶腺病毒
外文關鍵詞:NRIPMotor neuronGliosisChATAdenovirus
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核受體交互作用蛋白(Nuclear Receptor Interaction Protein, NRIP) 為本實驗室利用雄性激素受體(Androgen Receptor, AR) 的C端做餌,以yeast-two hybrid 選殖系統於human HeLa cDNA library所篩選而得。先前的研究發現,NRIP為AR的共同活化子以及是鈣離子訊息傳遞路徑中的一員,可透過NRIP結構上的IQ模組直接與攜鈣素(Calmodulin)結合並進一步活化鈣調磷酸酶(Calcineurin)以進行下游基因的轉錄調控。
鈣離子訊息傳遞路徑已在許多文獻中被證實其在神經生理及肌肉生理上皆扮演著重要的角色,在實驗室建立的NRIP基因全身剔除小鼠的動物模型研究中,NRIP knock-out小鼠在行為測驗中的滾輪測驗及跑步機測驗中表現出不正常的現象,而這樣的現象有可能是透過肌肉抑或神經的缺陷所造成。在先前的研究已發現,NRIP基因缺陷鼠的肌肉收縮強度及肌肉耐受性較正常老鼠差,但仍無法排除是否有運動神經缺陷的現象所共同影響,因此本篇論文旨在研究NRIP是否在運動神經元的運作中扮演部分功能。
根據脊髓冷凍切片並進行Nissl-staining 及乙醯膽鹼轉移酶(Choline Acetyltransferase, ChAT)免疫組織化學染色的結果,NRIP基因缺陷成鼠在腰部腹側脊髓的運動神經元有明顯的缺失,進一步利用免疫螢光染色發現其α運動神經元有缺陷的現象。同樣利用免疫螢光染色方式另外發現該處脊髓有常見的中樞神經系統受損的現象包括星狀神經膠質化(Astrogliosis)及微膠神經膠質化(Microgliosis)。由於在NRIP基因缺陷幼鼠中仍發現存在正常運動神經元的小鼠,因此這樣的結果應非發育時期所造成而可能是發育後與年齡相關的。而在細胞模型實驗中,NG108細胞株在不同分化時期利用即時聚合酶鏈式反應發現,NRIP及乙醯膽鹼轉移酶於該膽鹼能神經(Cholinergic neuron)中分化中皆被正向調控(up-regulated)。而為了進一步研究NRIP及其IQ結構的功能,我們已建構完成兩株腺病毒(Adenovirus)分別攜帶有NRIP及剔除IQ結構的NRIP,可於細胞株中進行外送基因的實驗或進而研究NRIP對ChAT基因的調控機轉。


NRIP, nuclear receptor interaction protein, is a novel gene we identified by using yeast-two hybrid system to prey a human HeLa cDNA library with the C terminal domain of androgen receptor as bait. NRIP was reported as an AR co-activator and is involved in Ca2+ signaling pathway which regulates many physiological functions including muscles and neuron by interaction with calmodulin through its IQ motif.
NRIP conventional knock-out (KO) mice show abnormal exercise performance on rota-rod and treadmill test. This phenotype may also relate to muscle and neuron functions. Our unpublished data show that NRIP plays a role in muscle strength and endurance performance. Now we investigated whether NRIP also played a role in motor neurons (MNs) functions.
By Nissl-staining and immunohistochemistry for choline acetyltransferase (ChAT), adult KO mice showed MNs loss in lumbar ventral horn spinal cord while immunofluorescence assay (IFA) stained the markers for α-MNs also showed deficient phenotype. IFA also showed that astrocyte and microglia were concentrated and activated in the region that is phenomena commonly occurred in central nervous system injury situation calls gliosis. These results might not be developmental but were age-related due to the KO mice could show normal phenotype in younger age. In the analysis of cholinergic neural cell line differentiation, NRIP and ChAT were both up-regulated in NG108 by quantitative RT-PCR. For further investigation the mechanism of NRIP regulating ChAT, we had success constructed adenoviruses carried NRIP or NRIPΔIQ for future experiment.


口試委員審定書 I
致謝 II
中文摘要 III
Abstract V
Chapter 1 INTRODUCTION 3
1.1 The characteristic of nuclear receptor interaction protein (NRIP). 3
1.2 Calcium signals in biological functions. 4
1.2.1 Calcium signals in neuron. 4
1.2.2 Calcium signals in muscle 6
1.3 The properties of motor neuron. 7
1.4 The development of motor neuron. 8
1.5 Aim of this study 9
Chapter 2 MATERIALS AND METHODS 11
2.1 Mice cohorts. 11
2.2 Western blot analysis. 11
2.3 Mice spinal cord isolation and the prepared of frozen section. 11
2.4 Double-labeled immunofluorescence assay. 12
2.5 Nissl-staining protocol. 13
2.6 Immunohistochemistry for frozen tissue sections. 13
2.7 Cell culture. 14
2.8 RNA isolation and quantitative real-time PCR. 14
2.9 Adenovirus construction. 15
2.9.1 Competent cell prepared protocol. 17
2.9.2 Ethanol precipitated plasmid isolation protocol. 18
2.9.3 Adenovirus genome DNA isolation protocol. 19
2.10 Statistical analysis. 19
Chapter 3 RESULTS 21
3.1 The colocalization of NRIP and ChAT in mice lumbar spinal cord. 21
3.2 The loss and the deficient of MNs in NRIP KO mice in spinal cord. 21
3.3 The gliosis in KO mice spinal cord. 23
3.4 NRIP can protect MNs due to age-related but not development. 25
3.5 The differentiation of cholinergic cell line NG108. 26
3.6 The construction of adenovirus-NRIP and adenovirus-NRIPΔIQ. 27
Chapter 4 DISCUSSION 29
Chapter 5 FIGURES 34
Chapter 6 REFERENCES 47
APPENDIX 51


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