臺灣博碩士論文加值系統

TMPRSS3是第一個被提出與非症候群聽障有關的絲胺酸蛋白。先前實驗室已經利用斑馬魚作為模式動物去探討與人類TMPRSS3的同源基因tmprss4a和tmprss4b的相關研究。同時也發現斑馬魚tmprss3的基因可能是人類TMPRSS3基因的同源基因。因此，本研究主要的目的是想進一步探討和瞭解斑馬魚tmprss3基因所扮演的角色。本研究首先利用生物資訊學的方法比對人類TMPRSS3和老鼠Tmprss3與斑馬魚tmprss3之關係。我們發現，兩個斑馬魚tmprss3a和tmprss3-like基因有可能和小鼠Tmprss3和人TMPRSS3基因同源。從半定量RT-PCR的結果去觀察tmprss3a和tmprss3-like在斑馬魚發育時期間的表現量，可以看到tmprss3a基因從胚胎受精後1.5小時就有微量的表現，直到受精後120小時皆有表現，其中在受精後25至120小時表現量有增加的趨勢。另外tmprss3-like則是從胚胎受精後1.5小時都有表現，但在受精後9至12小時表現相對較少。另外從半定量RT-PCR 的結果中，看到tmprss3a、tmprss3-like基因在斑馬魚成魚組織的表現看到tmprss3a在心臟和腎臟當中的表現量最高，其次為內耳；另外tmprss3-like則是在輸精管最高表現，其次是腎臟。在全胚胎原位雜交(Whole-mount in situ hybridization)中，隨著胚胎發育的時間越長，tmprss3a和tmprss3-like訊號慢慢的明顯，但tmprss3a在發育前期，表現則是較低的。tmprss3a和tmprss3-like在24小時是有表現在脊椎腹側。另一方面我們也利用顯微注射的方式打入反股的Morpholino（MO）去抑制tmprss3-like的轉譯，觀察到施打MO 4ng後pTaqRFP tmprss3-like 的螢光表現從85.67±0.08% 降至23±0.06%。另外，對於pTagRFP的螢光MO並不會造成影響，因此我們的結果可證明tmprss3-like Mo會專一性抑制tmprss3-like -pTaqRFP螢光表現。綜合以上所述，在本研究中我們雖然獲得tmprss3a和tmprss3-like在斑馬魚胚胎發育和組織內的表現情形，然而對於tmprss3a和tmprss3-like在斑馬魚上所扮演的角色仍需更進一步的探討。

Human TMPRSS3 which is a member of the Type II transmembrane serine protease (TTSP) was the first description of a serine protease involved in deafness. In our previous studies, we have used zebrafish as a model organism to explore tmprss4a and tmprss4b which are the homologue of human TMPRSS3. We also found that zebrafish tmprss3 may be a homologue of human TMPRSS3. The main purpose of this study is to further explore and understand the role of zebrafish tmprss3. We found that both zebrafish tmprss3a and tmprss3-like are probably homologous to human TMPRSS3 and mouse Tmprss3 through BLAST (Basic Local Alignment Search Tool). During embryogenesis, reverse transcription polymerase chain reaction (RT-PCR) revealed that zebrafish tmprss3a was expressed after 1.5 post-fertilization (hpf), and a relatively high level of expression was detected between 25-120 hpf. On the other hand, zebrafish tmprss3-like was expressed from 1.5 to 120 hours hpf, but a relatively low level of expression was detected between 9-12 hpf. In addition, both tmprss3a and tmprss3-like transcripts were observed in multiple adult tissues through RT-PCR. Moreover, the highest expression of tmprss3a were detected in heart, kidney, second is innear. On the other hand, the highest expression of tmprss3-like was in vas deferens, second is kidneys. Whole-mount in situ hybridization revealed that the signals of tmprss3a and tmprss3-like were gradually obvious during embryogenesis for longer. However, the signal of tmprss3a was relatively low in the early stage. tmprss3a and tmprss3-like are specially express in the ventral spine at 24-hour stage. In addition, we developed one specific tmprss3-like-targeting morpholino oligonucleotides (MOs) and established the fusion protein construct TagRFP tmprss3-like that was co-injected into embryos in the presence or absence of morpholinos. We further knocked-down tmprss3-like by MOs, and observed that the application of 4ng MO could decrease the expression of pTaqRFP tmprss3-like fluorescent from 85.67 ± 0.08% to 23 ± 0.06%. In addition, MO against pTagRFP fluorescent do not be affected, so our results may prove that tmprss3-like Mo would be specific knockdown the expression of pTaqRFP tmprss3-like fluorescence. In summary, although we contributes to the understanding of the specific expression and distribution of tmprss3a and tmprss3-like in the embryonic development and tissue, the specific roles of tmprss3a and tmprss3-like in the zebrafish still have be investigate.

目錄......................................................i
縮寫表...................................................ii
中文摘要..................................................1
英文摘要..................................................3
第一章 序論 （Introduction）...............................5
第二章 材料與方法 （Meterials and Methods）.................10
第三章 結果 （Results）...................................24
第四章 討論 （Discussion） ................................29
未來展望（Future work）...................................34
參考文獻 （References）..................................35
圖表 （Figures）...........................................38
附錄 （Appendixes） .....................................54

Ahmed, Z. M., X. C. Li, S. D. Powell, S. Riazuddin, T. L. Young, K. Ramzan, Z. Ahmad, S. Luscombe, K. Dhillon, L. MacLaren, B. Ploplis, L. I. Shotland, E. Ives, S. Riazuddin, T. B. Friedman, R. J. Morell and E. R. Wilcox (2004). "Characterization of a new full length TMPRSS3 isoform and identification of mutant alleles responsible for nonsyndromic recessive deafness in Newfoundland and Pakistan. BMC Med Genet ,5,24.
Brand, M., Heisenberg, CP., Jiang, YJ., Beuchle, D., Lun, K., Furutani-Seiki, M., Granato, M., Haffter, P., Hammerschmidt, M., Kane, DA., et al. Mutations in zebrafish genes affecting the formation of the boundary between midbrain and hindbrain. Development, 123,179-190.
Bouchard, M., P. Pfeffer and M. Busslinger (2000). Functional equivalence of the transcription factors Pax2 and Pax5 in mouse development. Development ,127, 3703-3713.
Corey, D. R. and J. M. Abrams (2001). Morpholino antisense oligonucleotides: tools for investigating vertebrate development. Genome Biol ,2, REVIEWS1015.
Couloigner, V., M. Fay, S. Djelidi, N. Farman, B. Escoubet, I. Runembert, O. Sterkers, G. Friedlander and E. Ferrary (2001). Location and function of the epithelial Na channel in the cochlea. Am J Physiol Renal Physiol ,280,F214-222.
Guipponi, M., G. Vuagniaux, M. Wattenhofer, K. Shibuya, M. Vazquez, L. Dougherty, N. Scamuffa, E. Guida, M. Okui, C. Rossier, M. Hancock, K. Buchet, A. Reymond, E. Hummler, P. L. Marzella, J. Kudoh, N. Shimizu, H. S. Scott, S. E. Antonarakis and B. C. Rossier (2002). The transmembrane serine protease (TMPRSS3) mutated in deafness DFNB8/10 activates the epithelial sodium channel (ENaC) in vitro. Hum Mol Genet ,11, 2829-2836.
Higgs, D. M., M. J. Souza, H. R. Wilkins, J. C. Presson and A. N. Popper (2002). Age- and size-related changes in the inner ear and hearing ability of the adult zebrafish (Danio rerio). J Assoc Res Otolaryngol ,3,174-184.
Hobson, J. P., S. Netzel-Arnett, R. Szabo, S. M. Rehault, F. C. Church, D. K. Strickland, D. A. Lawrence, T. M. Antalis and T. H. Bugge (2004). Mouse DESC1 is located within a cluster of seven DESC1-like genes and encodes a type II transmembrane serine protease that forms serpin inhibitory complexes. J Biol Chem ,279, 46981-46994.
Hooper, J. D., J. A. Clements, J. P. Quigley and T. M. Antalis (2001). Type II transmembrane serine proteases. Insights into an emerging class of cell surface proteolytic enzymes. J Biol Chem, 276,857-860.
Hooper, J. D., J. A. Clements, J. P. Quigley and T. M. Antalis (2001). Type II transmembrane serine proteases. Insights into an emerging class of cell surface proteolytic enzymes. J Biol Chem ,276,857-860.
Hu, Y. H., H. J. Warnatz, D. Vanhecke, F. Wagner, A. Fiebitz, S. Thamm, P. Kahlem, H. Lehrach, M. L. Yaspo and M. Janitz (2006). Cell array-based intracellular localization screening reveals novel functional features of human chromosome 21 proteins. BMC Genomics ,7,155.
Kimmel, C. B., W. W. Ballard, S. R. Kimmel, B. Ullmann and T. F. Schilling (1995). "Stages of embryonic development of the zebrafish. Dev Dyn, 203, 253-310.
Kinney, S. R. and S. Pradhan (2011). Regulation of expression and activity of DNA (cytosine-5) methyltransferases in mammalian cells. Prog Mol Biol Transl Sci ,101, 311-333.
Lee, J., J. I. Baek, J. Y. Choi, U. K. Kim, S. H. Lee and K. Y. Lee (2013). Genetic analysis of TMPRSS3 gene in the Korean population with autosomal recessive nonsyndromic hearing loss. Gene, 532,276-280.
Lieschke, G. J. and P. D. Currie (2007). Animal models of human disease: zebrafish swim into view. Nat Rev Genet ,8,353-367.
Ohler, A. and C. Becker-Pauly (2011). Morpholino knockdown of the ubiquitously expressed transmembrane serine protease TMPRSS4a in zebrafish embryos exhibits severe defects in organogenesis and cell adhesion. Biol Chem, 392, 653-664.
Robu, M. E., J. D. Larson, A. Nasevicius, S. Beiraghi, C. Brenner, S. A. Farber and S. C. Ekker (2007). p53 activation by knockdown technologies. PLoS Genet ,3,e78.
Szabo, R., S. Netzel-Arnett, J. P. Hobson, T. M. Antalis and T. H. Bugge (2005). Matriptase-3 is a novel phylogenetically preserved membrane-anchored serine protease with broad serpin reactivity. Biochem J ,390,231-242.
Terzic, J., C. Muller, S. Gajovic and M. Saraga-Babic (1998). Expression of PAX2 gene during human development. Int J Dev Biol, 42,701-707.
Timme-Laragy, A. R., S. I. Karchner and M. E. Hahn (2012). Gene knockdown by morpholino-modified oligonucleotides in the zebrafish (Danio rerio) model: applications for developmental toxicology. Methods Mol Biol, 889,51-71.
Torres, M., E. Gomez-Pardo, G. R. Dressler and P. Gruss (1995). Pax-2 controls multiple steps of urogenital development. Development, 121, 4057-4065.
Torres, M., E. Gomez-Pardo and P. Gruss (1996). Pax2 contributes to inner ear patterning and optic nerve trajectory. Development, 122,3381-3391.
Veske, A., R. Oehlmann, F. Younus, A. Mohyuddin, B. Muller-Myhsok, S. Q. Mehdi and A. Gal (1996). Autosomal recessive non-syndromic deafness locus (DFNB8) maps on chromosome 21q22 in a large consanguineous kindred from Pakistan. Hum Mol Genet, 5.
Warga, R. M. and C. B. Kimmel (1990). Cell movements during epiboly and gastrulation in zebrafish. Development, 108,569-580.
Whitfield, T. T., B. B. Riley, M. Y. Chiang and B. Phillips (2002). Development of the zebrafish inner ear. Dev Dyn ,223, 427-458.
王怡文 (2013) 斑馬魚tmprss基因之鑑定與功能分析.中山醫學大學生物醫學科學學系碩士班學位論文。
楊燦鴻(2011) TMPRSS3基因之功能分析.中山醫學大學生物醫學科學學系碩士班學位論文。