LMBRD1蛋白質具有467個胺基酸（約53kDa），經由序列分析，其第4至13個胺基酸可能會與肌動蛋白結合，而第193至209個胺基酸可能為核位訊號（nuclear localization signal; NLS）。由小鼠的實驗研究結果得知此LMBRD1蛋白質在老鼠實驗方面最主要作用於神經系統的海馬迴(Hippocampus)。LMBRD1缺失之小鼠，其後肢微跛，推測其肌肉發育可能受影響。為了深入研究LMBRD1如何在胚胎發育早期參與肌肉的發育，我們選用斑馬魚為材料，首先，利用野生種(WT)斑馬魚分別對各時期之胚胎執行原位雜交實驗，加以探討LMBRD1與斑馬魚早期胚胎肌肉發育時期之間的相互關係，研究結果得知其signal表現皆在頭部、嗅球、體節與肌肉，但是在脊索並無任何訊息表現。為了更近一步確認LMBRD1蛋白質表現位為肌肉組織，所以藉著LMBRD1執行抗體染色，觀察結果等同於原位雜交實驗結果。緊接著為了更加暸解NESI在斑馬魚之作用機轉，我們利用Knockdown LMBRD1方式，可觀察得到斑馬魚胚胎頭部萎縮以及魚體軀幹彎曲，緊接著再利用F59、phalloidin、α-actin、α-actinin、Dystrophinu一系列之肌肉與細胞骨架之標記抗體並染色觀察之，發現斑馬魚肌纖維排列混亂、崩解，肌動蛋白排列不規則，體節間隔板排列彎曲，肌束與肌束間隔變寬，嚴重者甚至可能造成運動神經軸索無法正常延伸發育以及心肌細胞呈現較為細長，未折疊完全之情形出現。經由F59抗體標記染色後再冷凍切片觀察得知其LMBRD1-MO之慢肌較WT排列不規則，慢肌數量上也有減少之趨勢；快肌則表現量也較少。由此，我們認為這新穎的LMBRD1蛋白質對斑馬魚胚胎在早期肌肉發育的形成與心臟發育具有關鍵性的角色。

LMBR1 domain containing 1 protein(LMBRD1),also known as NESI,is involved in a novel nuclear export pathway.Characteristic phenotypes of the heterozygous knockout mice include stumbling,lifting the hind-legs,and dragging tails during movement suggest that LMBRD1 plays significant roles in motor coordination,learning,and memory during neuronal differentiation.To further examine its biological functions during early embryogenesis,we used zebrafish as a model system because of their well-defined developmetal stages and genetic background.We stained the embryos with a peptide antibody against human LMBRD1(αhLMBRD1) to detect the spatiotemperal expression patterns of endogenous zebrafish LMBRD1 polypeptide.At 24 hpf,expression was detected throughout the somatic mesoderm in the both fast and slow muscle populations later,zebrafish LMBRD1 was restricted in the future retina,otic placode,brain,muscle and pectoral fin buds,but were down-regulated in notochord.Transverse section through the trunk of a 72 hpf embryo showing the zebrafish LMBRD1 express on the surface of each myofibril.In the addition,zebrafish LMBRD1 express at high level in retina ganglion and brain cortex but is down-regulated in the mature muscle of adult fish.By the way,also injected the LMBRD1-morpholino into zebrafish embryos to observe the difference.The LMBRD1 knockdown fish displayed reduced head and curved-body phenotypes.To further investigate the molecular mechanisms that lead to curved-body phenotypes,we stained embryos by some muscle specific markers,such as F59,phalloidin,α-actin and dystrophin,to detect the morphological changes in muscle fiber,interactions among cytoskeletal components and membrane,respectively.Our results show that the muscle fibers of LMBRD1-morphant aligned disorderly and lost their integrity,septum curved,misalignment of motor neuron innervations,the myocardium slender.Fast and slow muscle numbers are also less than those of WT.These data suggest that LMBRD1 may function to regulate muscle fiber organization and play an important role zebrafish myogenesis.

中文摘要---------------------------------Ⅰ
英文摘要---------------------------------Ⅱ
目錄-------------------------------------Ⅳ
圖表目錄---------------------------------VI



一、前言-------------------------------------01
1.1 胚胎早期肌肉發育-------------------------------	01
1.2 肌肉細胞的結構與收縮方式-----------------------	03
1.3 胚胎早期心臟肌肉的發育-------------------------	04
1.4 肌肉細胞蛋白質間的交互作用---------------------	05
1.5 肌肉細胞的神經傳動與鈣離子---------------------	06
1.6維生素B12 (氰鈷胺酸)生理功能---------------------	07
1.7 LMBRD1的發現與命名-----------------------------	07
1.8 LMBRD1與Cobalamin之間的相互關係---------------	08
1.9 LMBRD1 在小鼠體內的表現------------------------	09
1.10 斑馬魚的優勢----------------------------------	10


二、材料方法---------------------------------11
2.1.斑馬魚飼養及胚胎的收集-------------------------	12
2.2.RNA萃取---------------------------------------	12
2.3. LMBRD1 Primer的設計--------------------------	13
2.4.反轉錄酶-聚合酶鏈反應（RT-PCR）進行Zebrafish Lmbrd1之基因選殖------------------------------	
13
2.5.大腸桿菌轉型法（Transformation）---------------	14
2.6 .小量質體DNA之萃取----------------------------	14
2.7.探針(Riboprobe)合成----------------------------	16
2.8.原位雜交染色法---------------------------------	17
2.9.免疫抗體螢光染色-------------------------------	19
2.10.Phalloidin staining for F-actin---------------	20
2.11.顯微注射實驗----------------------------------	20
2.12 切片及樣品製備--------------------------------	21
2.13 序列比對--------------------------------------	21
2.14 設備------------------------------------------	21


三、結果-------------------------------------23
3.1 斑馬魚與其他脊椎動物的Lmbrd1 胺基酸序列比對與演化樹分析圖----------------------------------	
24
3.2 藉由斑馬魚之胚胎進行原位雜交法觀察LMBRD1之時空分佈------------------------------------------	
24
3.3 觀察斑馬魚胚胎之LMBRD1基因在蛋白質層面表現----	25
3.4 knock LMBRD1 down後所產生之LMBRD1 表現型態----	27
3.5 LMBRD1在斑馬魚體節肌肉生成方面之功能----------	28
3.6 LMBRD1 morpholino注射後的存活率以及肌肉缺失比率	29
3.7 Cobalamin是可補救LMBRD1 Knockdown所造成的缺陷	30
3.8 LMBRD1 與myoD在斑魚軀幹肌肉細胞生長上的相互關係	32
3.9 LMBRD1與肌肉的結構蛋白質標記(α-actin)的相互關係--------------------------------------------	
32
3.10 藉由dystrophin之免疫抗體染色法，來探討LMBRD1與細胞骨架之間的相互關係--------------------------	
33
3.11 LMBRD1 與斑馬魚胚胎心臟肌肉之相互作用--------	34


四、討論-------------------------------------36
4.1 脊椎動物LMBRD1之表現時期與位置----------------	37
4.2  抑制脊椎動物LMBRD1表現後所呈現的表現型態-----	38
4.3  LMBRD1在軀幹部位肌肉生長機制中所扮演的角色---	40
4.4 總結-------------------------------------------	41


五、參考文獻---------------------------------43
六、圖表-------------------------------------48
七、附錄-------------------------------------66

圖表目錄
Fig.01 LMBRD1的胺基酸序列比對以及演化樹分析圖。------------49
Fig.02 LMBRD1基因在胚胎發育各個時期之表現與空間分佈。------51
Fig.03觀察斑馬魚胚胎之LMBRD1基因之蛋白質表現。 -----------52
Fig.04螢光染色後切片之成魚之內部蛋白質表現。--------------54
Fig.05 knockdown LMBRD1後的LMBRD1變異表現型態。-----------55
Fig.06 knockdown LMBRD1 後斑馬魚肌肉細胞標記-F59表現。----56
Fig.07 F59與phalloidin在Lmbrd1 morphant肌肉表現情形。------57
Fig.08 myoD在斑馬魚mRNA肌肉表現訊號圖。------------------58
Fig.09α-actin在斑馬魚mRNA肌肉表現訊號圖。----------------59
Fig.10斑馬魚體節間隔板之變異圖。--------------------------60
Fig.11  F59在斑馬魚胚胎心肌之表現訊號圖-------------------61


Table.1 斑馬魚與其他脊椎動物Lmbrd1胺基酸序列相似性。------62
Table.2 LMBRD1 morpholino Cobalamin的存活率與表現率。-----63
Table.3 LMBRD1 morpholino 與野生種斑馬魚心跳次數統計/1分鐘-64
Table.4 Lmbrd1 morphant心肌細胞活動表現情形以及血循流動---65

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邱雅倫(民96)核輸出訊息結合蛋白質NESI之肌動蛋白結合區之功分析，國立台灣大學醫學院生物化學暨分子生物學研究所碩士論文