鈣離子的調控對於成體肌肉的影響是被大家所熟知的，例如咖啡因會促使鈣離子釋放進入細胞，促使肌肉細胞發生短期的痙攣,並造成肌肉痠痛及無力。但鈣離子的功能對於肌肉早期發育的影響如今並非如此明瞭。本論文利用2-APB (2-Aminoethoxydiphenyl borate)對於細胞內鈣離子濃度進行調控，進而觀察鈣離子對斑馬於早期肌肉發育所造成的影響。我們的實驗結果指出，在經過浸泡2-APB後之斑馬魚體內鈣離子的數量確實被抑制並使魚體產生彎曲，以及肌肉纖維變異等不正常表型。同時我們經由針對myod所做之原為雜交實驗，發現在浸泡2-APB後，會過度表現與表現位置異常，因此我們利用myod mopholino來降低魚體內其mRNA的表現量,再以2-APB浸泡，可以發現肌肉纖維回復正常的狀態。除此之外，在我們的實驗中，利用咖啡因來提升細胞內鈣離子的濃度，也確實可以挽救2-APB所導致之細胞內鈣離子不足所造成之不正常表型。為了更加確定對於斑馬於早期肌肉發育的調控是由鈣離子濃度所驅動,我們也針對了一些與鈣離子相關並參與肌肉發育和分化的基因做定量-PCR (real-time PCR) ，我們發現desma、fbxo32及nfatc1基因會因2-APB浸泡而表現量上升，反之，cpped1基因會因2-APB浸泡而表現量下降，利用這些證據，我們建立了一套模型，期望藉此得以說明鈣離子訊號在斑馬魚肌肉發育過程中所扮演的角色。

Calcium signaling plays important physiological roles on muscle physiology. For example, enhancement of cellular calcium concentration causes short-term spasm, then make muscle faintness or pain. However, the function of intracellular calcium signaling is unclear during early myogenesis. In this thesis, we used 2-APB(intracellular calcium blocker) and caffeine((intracellular calcium inducer) to regulate intracellular calcium concentration and to investigate the effect caused by calcium during early myogenesis. Our data showed that 2-APB treated zebrafish embryos displayed some malformed phenotypes, such as defective somite and myofribril misalignment. We also found that myod signals were up-regulated and expressed disorderly in the 2-APB-treated embryos. Knock down of myod expression can reverse 2-APB-induced muscle defect phenotypes. Results from real-time PCR showed that desma, fbxo32, and nfatc1 were up regulated but cpped1 was down regulated in 2-APB-treated embryos. On the basis of these observations, we proposed a model to elucidate how intracellular calcium delicate regulates early myogenesis

謝誌……………………………………………………………………I
中文摘要………………………………………………………………Ⅱ
英文摘要………………………………………………………………Ⅲ
目錄……………………………………………………………………Ⅳ
圖表目錄………………………………………………………………Ⅷ
第一章 前言…………………………………………………………1
第一節: 鈣對人體的重要性……………………………………………1
1.1 人體中的鈣…………………………………………………………1
1.2 人體中的鈣離子……………………………………………………1
1.3 鈣離子之調控與相關徵狀…………………………………………3
第二節: 肌肉與鈣離子…………………………………………………5
2.1 肌肉的組成…………………………………………………………5
2.2 肌肉發育……………………………………………………………5
2.3 肌肉與鈣離子關係…………………………………………………7
第三節: 模式物種-斑馬魚……………………………………………8
第四節: 研究動機………………………………………………………9
第二章 材料與方法………………………………………………102.1 斑馬魚飼養及胚胎的收集……………………………………10
2.2 顯微注射技術…………………………………………………10
2.3 鈣離子染劑分析………………………………………………12
2.4 藥物浸泡………………………………………………………13
2.5 甩尾能力記數 (in-chorion contraction)………………………15
2.6 免疫抗體染色…………………………………………………15
2.7 原位雜交反應(Whole-mount in situ hybridization)……………16
2.8 冷凍切片前處理及方法………………………………………20
2.9 蘇木紫與伊紅染色(H＆E staining)……………………………21
2.10 螢光定量聚合酶鍊鎖反應(Q-PCR) …………………………21
2.11 原位雜交反應藥品配製………………………………………23
2.12 設備與軟體……………………………………………………25
第三章 結果………………………………………………………26
3.1 2-APB浸泡條件與存活率………………………………………26
3.2 浸泡2-APB確實使魚體肌肉細胞內鈣離子量降低……………27
3.3 不同2-APB浸泡條件對斑馬魚胚胎產生之影響………………28
3.3.1 2-APB浸泡使得斑馬魚胚胎外表畸形……………………28
3.3.2 2-APB影響斑馬魚胚胎的運動能力………………………30
3.3.3 2-APB影響慢肌與肌動蛋白的排列………………………313.4 咖啡因使鈣離子濃度上升亦造成斑馬魚胚胎肌肉變異……32
3.4.1 咖啡因浸泡造成波浪狀肌纖維表現型………………………32
3.4.2 以咖啡因提升細胞內鈣離子濃度可挽救2-APB引起的細胞鈣離子濃度不足之表型………………………………………………33
3.5 2-APB影響myod表現………………………………………34
3.5.1 2-APB浸泡後導致myod表現異常…………………………34
3.5.2 降低myod表現量可減低因2-APB引起的不正常肌纖維表型……………………………………………………………………35
3.6 影響肌肉發育的相關基因……………………………………37
3.7 神經端鈣離子來源上升造成不同於2-APB的變異表型……39
3.7.1 D-絲氨酸造成波浪狀肌纖維表型……………………………39
3.7.2 DAO MO造成開闊肌纖維表型………………………………40
第四章 討論………………………………………………………41
4.1 2-APB使細胞內鈣離子濃度下降影響肌肉發育………………41
4.2 咖啡因使細胞內鈣離子濃度上升並影響肌肉發育……………42
4.3 嘗試利用咖啡因與2-APB調控肌肉發育………………………43
4.4 2-APB是否確實降低魚體內鈣離子濃度…………………………43
4.5 監測鈣離子對肌肉發育相關基因的影響………………………45
4.6 神經訊號鈣離子來源與未來方向………………………………47參考文獻………………………………………………………………49
圖表……………………………………………………………………53圖表目錄
Fig.1 2-APB浸泡方法與存活率………………………………………54
Fig.2鈣離子染劑螢光分析圖…………………………………………55
Fig.3 2-APB浸泡後魚體外型與組織切片……………………………56
Fig.4 2-APB浸泡後之運動能力………………………………………57
Fig.5 2-APB浸泡後慢肌與肌動蛋白的表現狀態……………………58
Fig.6咖啡因浸泡以及利用咖啡因挽救2-APB造成之影響…………59
Fig.7細胞內鈣離子濃度上升與下降造成不同肌纖維表現型示意圖60
Fig.8浸泡2-APB前後斑馬魚體內myod之空間分佈………………61
Fig.9 myod MO以及利用myod MO挽救2-APB造成之影響………62
Fig.10 增加細胞內鈣離子後肌纖維差異……………………………63
Fig.11 注射D-serine與DAO MO之變異率…………………………64
Table 1 利用不同濃度咖啡因挽救2-APB造成之表型………………65
Table 2 利用myod MO挽救2-APB造成之表型……………………66
Table 3定量PCR所使用的引子名稱與序列…………………………67
Table 4以1000 ppm咖啡因與10 uM 2-APB分別以方法II浸泡之定量PCR結果………………………………………………………………68
Table 5以500 ppm咖啡因與3 uM 2-APB分別以方法II浸泡之定量PCR結果………………………………………………………………69
Model 1 利用定量PCR之結果推估24 hpf時鈣離子的調控路徑………………………………………………………………………70

Asif, K.M., Paul M. K., Solomon, H. S. (2004). “D-Serine as a putative glial neurotransmitter.” Neuron Glia Biol. 1(3): 275–281.
Bertola, L.D., E. B. Ott, et al. (2008). “Developmental expression of the alpha-skeletal actin gene.” BMC Evol Biol 8: 166
Borello, U., M. Coletta, et al. (1999). “Transplacental delivery of the Wnt antagonist Frzb1 inhibits development of caudal paraxial mesoderm and skeletal myogenesis in mouse embryos.” Development 126(19): 4247-55
Burnet, P.W.J., Eastwood, S.L., Bristow, G.C., Godlewska, B.R., Sikka, P., Walker M., and Harrison, P.J. (2008). “D-Amino acid oxidase (DAO) activity and expression are increased in schizophrenia.” Mol Psychiatry. 13(7): 658–660. doi:10.1038/mp.2008.47.
Caroline, B., Maryam, M., Clare, E.F.D., and Rachel, A. (2005). “Acetylcholine and calcium signalling regulates muscle fibre formation in the zebrafish embryo.” Journal of Cell Science 118, 5181-5190.
Christoph, H., James, R., Jiandie, L., Paul, T., Tarr, Bruce, M., Spiegelman. (2003). “An autoregulatory loop controls peroxisome proliferator-activated receptor coactivator 1 expression in muscle.” PNAS ,vol. 100, no. 12, 7111–7116.
Dina, R., Elena, K., Maria, S., Chanda, M. C., Klinker, M. T., Bowser, and Herman, W. (2010). “Neuronal release of D-serine: a physiological pathway controlling extracellular D-serine concentration.” FASEB, Vol. 24, 0892-6638.
Donald, D., Ann, M., Rosaria, F., Catherine, L., Marc, P., Lutgarde, A., Michele, Z., and Serge, N. S. (2001). “Functional striatal hypodopaminergic activity in mice lackingadenosine A2A receptors.” Journal of Neurochemistry, 183-198.
Guyon, J. R., Mosley, A. N., hou, Y. Z, O‟Brien, K. F., Sheng, X., Chiang, K., Davidson, A. J., Volinski, J. M., Zon, L. I., and Kunkel, L. M.(2003). “The dystrophin associated protein complex in zebrafish.” Human Molecular Genetics, Vol. 12, No. 6, 601–615.
Jill, H., Clark, Nicholas, P., Kinnear, S. K., Gordon, C., Sidney, F., Loice, H., Jeyakumar, Frank W. A., Mark E. (2010). “Identification of Functionally Segregated Sarcoplasmic Reticulum Calcium Stores in Pulmonary Arterial Smooth Muscle.” JBC. M110.101485
John, G., McCarron, Marnie, L., Olson, Richard, D. R., Debbi, M., and Susan, C. (2007). “Ins(1,4,5)P3 receptor regulation during „quantal‟ Ca2+ release in smooth muscle.” TRENDS in Pharmacological Sciences Vol.28 No.6, 0165-6147.
Julie, L., Karen, C., Alfredo, C., Sabrina, B.P., Marie, P., Leibovitch, Serge, A., Leibovitch. (2009). “Inhibition of Atrogin-1/MAFbx Mediated MyoD Proteolysis Prevents Skeletal Muscle Atrophy In Vivo.” PLoS ONE vol. 4, issue 3, e4973.
Karen, W. L., Sarah, E. W., and Andrew, L. M. (2003). “Ca2+ released via IP3 receptors is required for furrow deepening during cytokinesis in zebrafish embryos.” Int. J. Dev. Biol. 47: 411-421.
Kun, H. L., Jae, Y. P., Kyungjin, K. (2004). “NMDA receptor-mediated calcium influx plays an essential role in myoblast fusion.” FEBS 578, 47–52.
Kunihiro, S., and Akihiko, Y. (2010). “The Functional Role of Calcineurin in Hypertrophy, Regeneration, and Disorders of SkeletalMuscle.” Journal of Biomedicine and Biotechnology vol. 2010, ID 721219, 8.
Matthew, J. B., and Andrew, R. M. (2010). “Ryanodine receptor channelopathies.” Pflugers Arch - Eur J Physiol DOI 10.1007.
Magdalena, B., Adam, S., Katarzyna, D., Marta, B. W., Aneta, K., Slawomir, P., Jacek, J., Jacek, K., Urszula, W. (2009). “Biochemical characterization and expression analysis of a novel EF-hand Ca2+ binding protein calmyrin2 (Cib2) in brain indicates its function in NMDAreceptor mediated Ca2+ signaling.” Biochemistry and Biophysics 487, 66–78.
Marco, S., Jiandie, L., Christoph, H., Wenli, Y., Zoltan, P. Arany, Stewart, H. Lecker, Alfred, L., Goldberg, and Bruce, M. S. (2006). “PGC-1α protects skeletal muscle from atrophy by suppressing FoxO3 action and atrophy-specific gene transcription.” PNAS, vol. 103, no. 44, 16260–16265.
Michael, J. B. (2006). “Calcium microdomains: Organization and function.” Cell Calcium vol. 40, 405–412
Nowicki, J.L. and A.C. Burke (2000). “Hox genes and morphological identity: axial versus lateral patterning in the vertebrate mesoderm.” Development 127(19): 4265-75
Peirang, C., Jun-ichi, H., Preeti, T., Shintaro, I., Vikas, P. Sukhatme, and Stewart, H. Lecker. (2009). “Statin-induced muscle damage and atrogin-1 induction is the result of a geranylgeranylation defect.” FASEB, Vol. 23, 0892-6638.
Pisaniello, A., C. Serra, et al. (2003). “The block of ryanodine receptors selectively inhibits fetal myoblast differentiation.” J Cell Sci 116(Pt8): 1589-97
Philip, E. Chen, Matthew, T. G., Elyse, K., Kevin, E., Matthew, R. L., Kate, K. O‟Toole, Phuong, L. C. Justin, L., James, P. Snyder, Stephen, F. Traynelis, and David, J. A.W. “Modulation of glycine potency in rat recombinant NMDA receptors containing chimeric NR2A/2D subunits expressed in Xenopus laevis oocytes.” J Physiol 586.1, 227–245.
Richard, D. R., John, G. M. (2009). “The sarcoplasmic reticulum Ca2+ store arrangement in vascular smooth muscle.” Cell Calcium 46 (2009) 313–322.
Timothy, A. McKinsey, Chun, L. Z., and Eric, N. O. (2000). “Activation of the myocyte enhancer factor-2 transcription factor bycalciumycalmodulindependent protein kinase-stimulated binding of 14-3-3 to histone deacetylase .” PNAS, vol. 97, no. 26, 14400–14405.
Yaniv, H., Daniel, P. S. O., and Simon, M. H. (2009). “Differential requirements for myogenic regulatory factors distinguish medial and lateral somitic, cranial and fin muscle fibre populations.” Development 136, 403-414 doi:10.1242/dev.028019.
吳欣儒(民98) LMBRD1參與斑馬魚肌肉發育之分子機轉探討 淡江大學碩士論文
吳嘉容(民94) 電生理活性與斑馬魚那離子通道1.6亞型在斑馬魚神經及肌肉發育上扮演的角色研究 國立陽明大學碩士論文
黃弈惠(民96) 咖啡因在斑馬魚早期胚胎發育時期引發之畸形 淡江大學碩士論文