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研究生: 葉雅欣
Yeh, Ya-Hsin
論文名稱: CaSR與PMCA1a在斑馬魚側線毛細胞上扮演之功能
Functional study of CaSR and PMCA1a in zebrafish lateral line hair cells
指導教授: 林豊益
Lin, Li-Yih
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2015
畢業學年度: 103
語文別: 中文
論文頁數: 49
中文關鍵詞: 毛細胞機械性傳導通道鈣離子平衡鈣離子感應接受器鈣離子通道
英文關鍵詞: Hair cell, MET channel, Calcium homeostasis, CaSR, PMCA1a
論文種類: 學術論文
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  • 哺乳動物的內耳毛細胞扮演偵測聲音的功能,當音波的震動傳入內耳,會使纖毛束彎曲並進而開啟機械性通道產生聽覺。毛細胞的機械性通道位於纖毛的頂端是一種對鈣離子具有高通透性的陽離子通道。近年來,斑馬魚的側線系統被廣泛研究,且側線毛細胞被做為活體研究的新指標。本篇研究利用掃描式離子選擇電極技術(SIET),以活體的狀況下測量斑馬魚仔魚側線毛細胞機械性通道的功能,進一步觀察PMCA與CaSR在毛細胞的表現以及對機械性通道功能的影響。在本篇實驗中證實PMCA1a與CaSR的蛋白質表現在受精後第96小時斑馬魚仔魚毛細胞靜纖毛的位置。以Morpholino knock down、PMCA抑制劑 (o-vanadate, Eosin-Y)、CaSR促進劑(R568)處理,發現機械性通道的鈣離子流受到抑制,表明了PMCA1a與CaSR對於機械性通道功能的重要性。此外,給予仔魚短期高鈣的環境也會抑制機械性通道鈣離子的流入,顯示環境鈣離子變化時機械性通道的功能會受到調節,而調節機械性通道的功能可能與PMCA1a以及CaSR參與有關。

    The sensory hair cells in mammalian inner ear are responsible for sound transduction. Sound waves deflect hair bundles and open the mechanotransducer (MET) channels, which initiate mechanotransduction. The MET channels expressed in stereocilia are cation channels with high Ca2+ permeability. Zebrafish lateral line is recently reported to be a useful in vivo model for studying hair cells. We have demonstrated the scanning ion-electrode technique (SIET) to be a sensitive approach for functionally assaying MET channel in zebrafish. In this study, expression and function of plasma membrane Ca2+ ATPase (PMCA) and calcium-sensing receptor (CaSR) were investigated. The protein expressions of PMCA1a and CaSR were showed in stereocilia of lateral line hair cell of 96 hpf zebrafish larvae. The MET channel mediated Ca2+ influx detected by SIET was suppressed by treatment of PMCA inhibitor (o-vanadate, Eosin Y) and CaSR activator (R568). These results indicated that PMCA1a and CaSR was critical for maintaining the function of MET channel. Furthermore, function of MET channel was decreased in larvae incubated in high Ca2+ water. These results revealed that function of MET channel is altered in different environmental Ca2+ levels. This functional regulation of MET channel may be required the participation of PMCA1a and CaSR.

    摘要 4 Abstract 5 研究背景 6 毛細胞(Hair cell) 6 機械性通道(Mechanoelectrical transducer (MET) channel) 6 Plasma membrane Ca2+-ATPase(PMCA) 7 Calcium-sensing receptor(CaSR) 8 斑馬魚的優勢與側線系統(Advantage of zebrafish and lateral-line system) 10 研究目的 12 材料方法 13 實驗動物(Animal) 13 人工淡水製備與馴養(Artificial water and acclimation) 13 掃描式離子選擇電生理技術(Scanning ion-selective electrode technique, SIET) 14 毛細胞離子流量測定(Measurement of hair cell calcium flux) 14 顯微注射與反義核酸(microinjection and antisense morpholino oligonucleotide, MO) 16 藥品製備與處理方法(Treatment with inhibitor/activator) 17 免疫組織化學染色(immunohistochemistry, IHC) 17 實驗流程 19 實驗設計 21 結果 23 實驗1-1:PMCA1a的蛋白質表現位置 23 實驗1-2:PMCA抑制劑(o-vanadate、Eosin-Y)對於MET channel功能的影響 23 實驗1-3:注射PMCA1a專一性反義核酸對於毛細胞的影響 24 實驗2-1:CaSR的蛋白質表現位置 25 實驗2-2:處理CaSR專一性促進劑(R568)對於MET channel功能的影響 25 實驗2-3:注射CaSR專一性反義核酸對於毛細胞的影響 26 實驗2-4:環境水體的鈣離子濃度變化對於MET channel功能的影響 26 討論 28 斑馬魚毛細胞的研究 28 PMCA在哺乳類毛細胞的研究 29 PMCA在魚類毛細胞的研究 30 CaSR在腎臟扮演的角色 31 CaSR在魚類的研究 31 CaSR的活性位置 33 總結 34 參考文獻 35 圖表 40 圖一:斑馬魚仔魚受精後第四天,PMCA免疫組織化學染色圖 40 圖二:PMCA抑制劑對斑馬魚側線毛細胞鈣離子流的變化 41 圖三:PMCA1a基因弱化後,對於斑馬魚仔魚的影響 42 圖四:PMCA1a基因弱化後,側線毛細胞鈣離子流的影響 43 圖五:斑馬魚仔魚受精後第四天,CaSR免疫組織化學染色圖 44 圖六:CaSR促進劑對斑馬魚側線毛細胞鈣離子流的變化 45 圖七:CaSR基因弱化後,對於斑馬魚仔魚的影響 46 圖八:水中鈣離子濃度變化對側線毛細胞鈣離子流的變化 47 圖九:CaSR基因弱化後,側線毛細胞鈣離子流的變化 48

    Alfadda, T.I., Saleh, A.M., Houillier, P. & Geibel, J.P. (2014) Calcium-sensing receptor 20 years later. American journal of physiology. Cell physiology, 307, C221-231.

    Amarjargal, N., Mazurek, B., Haupt, H., Andreeva, N., Fuchs, J. & Gross, J. (2008) Effects of SERCA and PMCA inhibitors on the survival of rat cochlear hair cells during ischemia in vitro. Physiological research / Academia Scientiarum Bohemoslovaca, 57, 631-638.

    Beurg, M., Nam, J.H., Chen, Q. & Fettiplace, R. (2010) Calcium balance and mechanotransduction in rat cochlear hair cells. Journal of neurophysiology, 104, 18-34.

    Bortolozzi, M., Brini, M., Parkinson, N., Crispino, G., Scimemi, P., De Siati, R.D., Di Leva, F., Parker, A., Ortolano, S., Arslan, E., Brown, S.D., Carafoli, E. & Mammano, F. (2010) The novel PMCA2 pump mutation Tommy impairs cytosolic calcium clearance in hair cells and links to deafness in mice. The Journal of biological chemistry, 285, 37693-37703.

    Chiu, L.L., Cunningham, L.L., Raible, D.W., Rubel, E.W. & Ou, H.C. (2008) Using the zebrafish lateral line to screen for ototoxicity. Journal of the Association for Research in Otolaryngology : JARO, 9, 178-190.

    Cruz, S., Shiao, J.C., Liao, B.K., Huang, C.J. & Hwang, P.P. (2009) Plasma membrane calcium ATPase required for semicircular canal formation and otolith growth in the zebrafish inner ear. The Journal of experimental biology, 212, 639-647.

    Donini, A. & O'Donnell, M.J. (2005) Analysis of Na+, Cl-, K+, H+ and NH4+ concentration gradients adjacent to the surface of anal papillae of the mosquito Aedes aegypti: application of self-referencing ion-selective microelectrodes. The Journal of experimental biology, 208, 603-610.

    Dumont, R.A., Lins, U., Filoteo, A.G., Penniston, J.T., Kachar, B. & Gillespie, P.G. (2001) Plasma membrane Ca2+-ATPase isoform 2a is the PMCA of hair bundles. The Journal of neuroscience : the official journal of the Society for Neuroscience, 21, 5066-5078.

    Esterberg, R., Hailey, D.W., Coffin, A.B., Raible, D.W. & Rubel, E.W. (2013) Disruption of intracellular calcium regulation is integral to aminoglycoside-induced hair cell death. The Journal of neuroscience : the official journal of the Society for Neuroscience, 33, 7513-7525.

    Feldman, B., Tuchman, M. & Caldovic, L. (2014) A zebrafish model of hyperammonemia. Molecular genetics and metabolism, 113, 142-147.

    Fettiplace, R. (2009) Defining features of the hair cell mechanoelectrical transducer channel. Pflugers Archiv : European journal of physiology, 458, 1115-1123.

    Ficarella, R., Di Leva, F., Bortolozzi, M., Ortolano, S., Donaudy, F., Petrillo, M., Melchionda, S., Lelli, A., Domi, T., Fedrizzi, L., Lim, D., Shull, G.E., Gasparini, P., Brini, M., Mammano, F. & Carafoli, E. (2007) A functional study of plasma-membrane calcium-pump isoform 2 mutants causing digenic deafness. Proceedings of the National Academy of Sciences of the United States of America, 104, 1516-1521.

    Germana, A., Abbate, F., Gonzalez-Martinez, T., del Valle, M.E., de Carlos, F., Germana, G. & Vega, J.A. (2004) S100 protein is a useful and specific marker for hair cells of the lateral line system in postembryonic zebrafish. Neuroscience letters, 365, 186-189.

    Ghysen, A. & Dambly-Chaudiere, C. (2007) The lateral line microcosmos. Genes & development, 21, 2118-2130.

    Go, W., Bessarab, D. & Korzh, V. (2010) atp2b1a regulates Ca2+ export during differentiation and regeneration of mechanosensory hair cells in zebrafish. Cell calcium, 48, 302-313.

    Hwang, P.P. & Chou, M.Y. (2013) Zebrafish as an animal model to study ion homeostasis. Pflugers Archiv : European journal of physiology, 465, 1233-1247.

    Jensen, T.P., Buckby, L.E. & Empson, R.M. (2004) Expression of plasma membrane Ca2+ ATPase family members and associated synaptic proteins in acute and cultured organotypic hippocampal slices from rat. Brain research. Developmental brain research, 152, 129-136.

    Jensen, T.P., Filoteo, A.G., Knopfel, T. & Empson, R.M. (2007) Presynaptic plasma membrane Ca2+ ATPase isoform 2a regulates excitatory synaptic transmission in rat hippocampal CA3. The Journal of physiology, 579, 85-99.

    Kozel, P.J., Davis, R.R., Krieg, E.F., Shull, G.E. & Erway, L.C. (2002) Deficiency in plasma membrane calcium ATPase isoform 2 increases susceptibility to noise-induced hearing loss in mice. Hearing research, 164, 231-239.

    Kwong, R.W., Auprix, D. & Perry, S.F. (2014) Involvement of the calcium-sensing receptor in calcium homeostasis in larval zebrafish exposed to low environmental calcium. American journal of physiology. Regulatory, integrative and comparative physiology, 306, R211-221.

    Liao, B.K., Deng, A.N., Chen, S.C., Chou, M.Y. & Hwang, P.P. (2007) Expression and water calcium dependence of calcium transporter isoforms in zebrafish gill mitochondrion-rich cells. BMC genomics, 8, 354.

    Lieschke, G.J. & Currie, P.D. (2007) Animal models of human disease: zebrafish swim into view. Nature reviews. Genetics, 8, 353-367.

    Lin, C.H., Su, C.H. & Hwang, P.P. (2014) Calcium-sensing receptor mediates Ca2+ homeostasis by modulating expression of PTH and stanniocalcin. Endocrinology, 155, 56-67.

    Lin, C.Y., Huang, C.C., Wang, W.D., Hsiao, C.D., Cheng, C.F., Wu, Y.T., Lu, Y.F. & Hwang, S.P. (2013a) Low temperature mitigates cardia bifida in zebrafish embryos. PloS one, 8, e69788.

    Lin, L.Y., Pang, W., Chuang, W.M., Hung, G.Y., Lin, Y.H. & Horng, J.L. (2013b) Extracellular Ca2+ and Mg2+ modulate aminoglycoside blockade of mechanotransducer channel-mediated Ca2+ entry in zebrafish hair cells: an in vivo study with the SIET. American journal of physiology. Cell physiology, 305, C1060-1068.

    Lin, Y.H., Hung, G.Y., Wu, L.C., Chen, S.W., Lin, L.Y. & Horng, J.L. (2015) Anion exchanger 1b in stereocilia is required for the functioning of mechanotransducer channels in lateral-line hair cells of zebrafish. PloS one, 10, e0117041.

    Loretz, C.A. (2008) Extracellular calcium-sensing receptors in fishes. Comparative biochemistry and physiology. Part A, Molecular & integrative physiology, 149, 225-245.

    Penheiter, A.R., Filoteo, A.G., Croy, C.L. & Penniston, J.T. (2001) Characterization of the deafwaddler mutant of the rat plasma membrane calcium-ATPase 2. Hearing research, 162, 19-28.

    Riccardi, D. & Kemp, P.J. (2012) The calcium-sensing receptor beyond extracellular calcium homeostasis: conception, development, adult physiology, and disease. Annual review of physiology, 74, 271-297.

    Schultz, J.M., Yang, Y., Caride, A.J., Filoteo, A.G., Penheiter, A.R., Lagziel, A., Morell, R.J., Mohiddin, S.A., Fananapazir, L., Madeo, A.C., Penniston, J.T. & Griffith, A.J. (2005) Modification of human hearing loss by plasma-membrane calcium pump PMCA2. The New England journal of medicine, 352, 1557-1564.

    Stauffer, T.P., Guerini, D. & Carafoli, E. (1995) Tissue distribution of the four gene products of the plasma membrane Ca2+ pump. A study using specific antibodies. The Journal of biological chemistry, 270, 12184-12190.

    Stawicki, T.M., Esterberg, R., Hailey, D.W., Raible, D.W. & Rubel, E.W. (2015) Using the zebrafish lateral line to uncover novel mechanisms of action and prevention in drug-induced hair cell death. Frontiers in cellular neuroscience, 9, 46.

    Street, V.A., McKee-Johnson, J.W., Fonseca, R.C., Tempel, B.L. & Noben-Trauth, K. (1998) Mutations in a plasma membrane Ca2+-ATPase gene cause deafness in deafwaddler mice. Nature genetics, 19, 390-394.

    Talarico, E.F., Jr., Kennedy, B.G., Marfurt, C.F., Loeffler, K.U. & Mangini, N.J. (2005) Expression and immunolocalization of plasma membrane calcium ATPase isoforms in human corneal epithelium. Molecular vision, 11, 169-178.

    Wada, M., Furuya, Y., Sakiyama, J., Kobayashi, N., Miyata, S., Ishii, H. & Nagano, N. (1997) The calcimimetic compound NPS R-568 suppresses parathyroid cell proliferation in rats with renal insufficiency. Control of parathyroid cell growth via a calcium receptor. The Journal of clinical investigation, 100, 2977-2983.

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