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Title: | 探討肌肉表現之NRIP在運動神經元退化中的角色 The role of muscle-restricted expression of NRIP in motor neuron degeneration |
Authors: | Kuan-Yu Liao 廖冠渝 |
Advisor: | 陳小梨(Show-Li Chen) |
Keyword: | 核受體交互作用蛋白,運動神經元,神經肌肉接點,肌肉表現因子,成肌素, NRIP,Motor neuron survival,Neuromuscular junction,Muscle-derived factors,myogenin, |
Publication Year : | 2015 |
Degree: | 碩士 |
Abstract: | 核受體交互作用蛋白(Nuclear receptor interaction protein, NRIP)是本實驗室在2005年以雄性激素受體(Androgen receptor)作為餌,從human HeLa cDNA library中利用酵母菌雙雜合(yeast-two hybrid)選殖系統篩選而得。我們發現NRIP可作為轉錄調節上的共同活化子去調控AR下游基因的表現,另外,NRIP也可在鈣離子存在情況下藉由與攜鈣素(calmodulin)之結合去調節肌肉的收縮運動。
從我們先前的研究中發現全身性NRIP基因剃除小鼠與正常小鼠相比,其肌肉強度較弱,且適應性運動行為表現也較差,此外,全身性NRIP基因剃除小鼠其腰部腹側脊髓的運動神經元也有缺失的現象。由於肌肉與運動神經元之間的關係十分緊密,能夠互相調控以維持正常的神經傳導及肌肉運動功能,另外,有文獻指出由肌肉所表現的一些因子能影響運動神經元的存活,因此在本篇論文中,我們想探討由肌肉所表現的NRIP對於運動神經元的存活所扮演的角色為何。 首先我們做出一隻肌肉NRIP基因剃除小鼠,其NRIP表現只會在肌肉組織中被剃除,肌肉以外的組織NRIP皆可正常表現。藉由免疫螢光染色發現肌肉NRIP基因剃除小鼠腰部腹側脊髓的運動神經元有缺失的現象,另外藉由全標本包埋染色法(whole-mounting staining)也發現肌肉NRIP基因剃除小鼠的神經肌肉接點(neuromuscular junction)有受到破壞的情形,代表肌肉表現的NRIP在維持運動神經元的存活以及神經肌肉接點的型態完整度中扮演著重要的角色。從行為實驗中,我們也進一步發現與正常小鼠相比,肌肉NRIP基因剃除小鼠表現出較差的運動能力。最後,我們想要探討肌肉是否會藉由NRIP去調控一些特殊因子的表現進而去調節神經肌肉接點的型態完整度以及運動神經元的存活,我們發現在肌肉NRIP基因剃除小鼠的比目魚肌中,成肌素(myogenin)的mRNA表現量會下降,而在感染Ad-shNRIP的C2C12細胞中,成肌素的mRNA表現也有下降的趨勢,這代表著NRIP的缺失會影響成肌素的表現量,因此我們認為在肌肉組織中NRIP能夠調控成肌素的表現量,並且,由肌肉所表現的成肌素在調控神經肌肉接點的型態完整度以及動神經元的存活可能扮演著重要的角色。 Nuclear receptor interaction protein (NRIP) is a novel nuclear receptor binding protein we identified by using AR as the bait to screen a human HeLa cDNA library in a yeast two-hybrid screening assay in 2005. NRIP functions as a transcriptional co-activator which mediates AR-driven gene expression and as a calcium-dependent calmodulin (CaM)-binding protein involved in muscle contraction. From our previous study, we found conventional NRIP knockout mice have reduced muscle force and impaired adaptive exercise performance. Lumbar motor neuron loss in spinal cord was also observed in conventional NRIP knockout mice. Previous research suggested that muscle-expressing factors may regulate motor neuron (MN) survival and maintaining. Thus, we want to investigate the role of muscle-specific expression of NRIP in motor neuron survival. First, we generated muscle-specific NRIP knockout mice, also named conditional KO (cKO) mice, as our experimental models. Results of immunofluorescence assay (IFA) and whole-mounting staining assay showed that cKO mice have less α-MNs and impaired neuromuscular junction (NMJ) in comparison to aged-matched WT mice, respectively. It implied that NRIP expressed by muscle plays an important role in maintaining motor neuron survival and NMJ integrity. From rotarod test, we also found cKO mice display deficient motor function compared with WT mice. Finally, we screened the muscle-derived candidate factor(s) which is capable of affecting motor neuron survival and NMJ integrity by performing RT-qPCR reaction. We found that mRNA levels of myogenin which is a myogenic transcription factor are down-regulated both in soleus of cKO mice and in C2C12 cell infected with Ad-shNRIP. These results implied that NRIP may regulate the expression of myogenin and myogenin may be the muscle-derived factor that can affect motor neuron survival and NMJ integrity. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/4681 |
Fulltext Rights: | 同意授權(全球公開) |
Appears in Collections: | 微生物學科所 |
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