神經網路的形成是腦雌雄性別分化的關鍵，神經的發育決定神經網路的型塑。本研究室以莫三比克口孵吳郭魚(Oreochromis mossambicus) 為探討腦性別分化(brain sexual differentiation) 的模式動物。本研究室已建立吳郭魚腦發育初期的表現基因資料庫 (Expressed Sequence Tags, ESTs)。本論文針對其中一條神經發育相關的基因，ptn (pleiotrophin)完成基因定序，並以原核生物表現系統進行PTN的蛋白質表現，以E. coli量產，經6X-His tag column純化後取得PTN，進一步，探討PTN對於神經細胞分化與增生的影響。另一方面，以老鼠神經母瘤細胞(Neuro-2a, N2a)為實驗模式細胞，應用轉染技術 (Transfection) ，轉殖綠色螢光蛋白 (green fluorescent protein,GFP) 基因，觀察PTN蛋白質在N2a細胞中分佈的位置。

The unique gene, pleiotrophin (ptn) was identified in the expressed sequence taq (EST) derived from the developing tilapia brain in our lab. The cDNA full length of ptn was cloned. ptn play a role in the differentiation of nerve cell. In this study, bioinformatics were searched for EE723939.1 (ptn), which is a gene with 1026 bp of cDNA sequence, open reading frame(ORF) is 483bp, and deduced as 160 amino acids. The protein of PTN was expressed in the prokaryotic system, BL21(E.coli), and purified with Ni-NTA affinity chromatography. In the present studies, ptn, cloned from tilapia, Oreochromis mossambicus. The influence of ptn on the proliferation of Neuro-2a cell was also investigated.The ORF of ptn was cloned, and the pEGFP-ptn plasmid was constructed. The distribution of ptn in the pEGFP-ptn transfected Neuro-2a cell was identified by fluorescence and laser confocal microscopy.

目次
致謝.............................................................i
中文摘要.....................................................ii
英文摘要.....................................................iii
前言.............................................................1
材料與方法.................................................3
結果............................................................27
討論............................................................30
參考文獻....................................................33
圖次............................................................38

Brannvall, K., Korhonen, L., & Lindholm, D. (2002). Estrogen-receptor-dependent regulation of neural stem cell proliferation and differentiation. [Research Support, Non-U.S. Gov't]. Mol Cell Neurosci, 21(3), 512-520.
Carrer, H. F., & Cambiasso, M. J. (2002). Sexual differentiation of the brain: genes, estrogen, and neurotrophic factors. [Research Support, Non-U.S. Gov't Review]. Cell Mol Neurobiol, 22(5-6), 479-500.
Chang, M. H., Huang, C. J., Hwang, S. P., Lu, I. C., Lin, C. M., Kuo, T. F., & Chou, C. M. (2004). Zebrafish heparin-binding neurotrophic factor enhances neurite outgrowth during its development. [Research Support, Non-U.S. Gov't]. Biochem Biophys Res Commun, 321(2), 502-509. doi: 10.1016/j.bbrc.2004.06.172
Clamp, M., Andrews, D., Barker, D., Bevan, P., Cameron, G., Chen, Y., . . . Birney, E. (2003). Ensembl 2002: accommodating comparative genomics. [Research Support, Non-U.S. Gov't]. Nucleic Acids Res, 31(1), 38-42.
Diaz-Hernandez, M., del Puerto, A., Diaz-Hernandez, J. I., Diez-Zaera, M., Lucas, J. J., Garrido, J. J., & Miras-Portugal, M. T. (2008). Inhibition of the ATP-gated P2X7 receptor promotes axonal growth and branching in cultured hippocampal neurons. [Research Support, Non-U.S. Gov't]. J Cell Sci, 121(Pt 22), 3717-3728. doi: 10.1242/jcs.034082
Herradon, G., & Ezquerra, L. (2009). Blocking receptor protein tyrosine phosphatase beta/zeta: a potential therapeutic strategy for Parkinson's disease. [Research Support, Non-U.S. Gov't Review]. Curr Med Chem, 16(25), 3322-3329.
Hienola, A., Pekkanen, M., Raulo, E., Vanttola, P., & Rauvala, H. (2004). HB-GAM inhibits proliferation and enhances differentiation of neural stem cells. [Research Support, Non-U.S. Gov't]. Mol Cell Neurosci, 26(1), 75-88. doi: 10.1016/j.mcn.2004.01.018
Kinnunen, T., Kaksonen, M., Saarinen, J., Kalkkinen, N., Peng, H. B., & Rauvala, H. (1998). Cortactin-Src kinase signaling pathway is involved in N-syndecan-dependent neurite outgrowth. [Research Support, Non-U.S. Gov't]. J Biol Chem, 273(17), 10702-10708.
Lauri, S. E., Kaukinen, S., Kinnunen, T., Ylinen, A., Imai, S., Kaila, K., . . . Rauvala, H. (1999). Reg1ulatory role and molecular interactions of a cell-surface heparan sulfate proteoglycan (N-syndecan) in hippocampal long-term potentiation. [In Vitro Research Support, Non-U.S. Gov't]. J Neurosci, 19(4), 1226-1235.
Li, Y. S., Milner, P. G., Chauhan, A. K., Watson, M. A., Hoffman, R. M., Kodner, C. M., . . . Deuel, T. F. (1990). Cloning and expression of a developmentally regulated protein that induces mitogenic and neurite outgrowth activity. [Comparative Study Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.]. Science, 250(4988), 1690-1694.
Lima, T., Auchincloss, A. H., Coudert, E., Keller, G., Michoud, K., Rivoire, C., . . . Bairoch, A. (2009). HAMAP: a database of completely sequenced microbial proteome sets and manually curated microbial protein families in UniProtKB/Swiss-Prot. [Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't]. Nucleic Acids Res, 37(Database issue), D471-478. doi: 10.1093/nar/gkn661
Marchionini, D. M., Lehrmann, E., Chu, Y., He, B., Sortwell, C. E., Becker, K. G., . . . Collier, T. J. (2007). Role of heparin binding growth factors in nigrostriatal dopamine system development and Parkinson's disease. [Comparative Study Research Support, N.I.H., Extramural]. Brain Res, 1147, 77-88. doi: 10.1016/j.brainres.2007.02.028
Meng, K., Rodriguez-Pena, A., Dimitrov, T., Chen, W., Yamin, M., Noda, M., & Deuel, T. F. (2000). Pleiotrophin signals increased tyrosine phosphorylation of beta beta-catenin through inactivation of the intrinsic catalytic activity of the receptor-type protein tyrosine phosphatase beta/zeta. Proc Natl Acad Sci U S A, 97(6), 2603-2608. doi: 10.1073/pnas.020487997
Oo, T. F., Marchionini, D. M., Yarygina, O., O'Leary, P. D., Hughes, R. A., Kholodilov, N., & Burke, R. E. (2009). Brain-derived neurotrophic factor regulates early postnatal developmental cell death of dopamine neurons of the substantia nigra in vivo. [Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't]. Mol Cell Neurosci, 41(4), 440-447. doi: 10.1016/j.mcn.2009.04.009
Papadimitriou, E., Mikelis, C., Lampropoulou, E., Koutsioumpa, M., Theochari, K., Tsirmoula, S., . . . Boudouris, P. (2009). Roles of pleiotrophin in tumor growth and angiogenesis. [Review]. Eur Cytokine Netw, 20(4), 180-190. doi: 10.1684/ecn.2009.0172
Perez-Pinera, P., Chang, Y., & Deuel, T. F. (2007). Pleiotrophin, a multifunctional tumor promoter through induction of tumor angiogenesis, remodeling of the tumor microenvironment, and activation of stromal fibroblasts. [Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.Review]. Cell Cycle, 6(23), 2877-2883.
Rauvala, H., Vanhala, A., Castren, E., Nolo, R., Raulo, E., Merenmies, J., & Panula, P. (1994). Expression of HB-GAM (heparin-binding growth-associated molecules) in the pathways of developing axonal processes in vivo and neurite outgrowth in vitro induced by HB-GAM. [Research Support, Non-U.S. Gov't]. Brain Res Dev Brain Res, 79(2), 157-176.
Shiue, Y. L., Wang, L. H., Chao, T. Y., Lin, C. H., & Tsai, C. L. (2004). EST-based identification of genes expressed in the hypothalamus of adult tilapia, Oreochromis mossambicus. [Research Support, Non-U.S. Gov't]. Biochem Biophys Res Commun, 316(2), 523-527. doi: 10.1016/j.bbrc.2004.02.079
Toran-Allerand, C. D. (2004). Estrogen and the brain: beyond ER-alpha and ER-beta. [Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.Review]. Exp Gerontol, 39(11-12), 1579-1586. doi: 10.1016/j.exger.2004.05.006
Tsai, C. L., Wang, L. H., Shiue, Y. L., & Chao, T. Y. (2007). Influence of temperature on the ontogenetic expression of neural development-related genes from developing tilapia brain expressed sequence tags. [Research Support, Non-U.S. Gov't]. Mar Biotechnol (NY), 9(2), 243-261. doi: 10.1007/s10126-006-6089-2
White, A. P., Collinson, S. K., Banser, P. A., Gibson, D. L., Paetzel, M., Strynadka, N. C., & Kay, W. W. (2001). Structure and characterization of AgfB from Salmonella enteritidis thin aggregative fimbriae. [Research Support, Non-U.S. Gov't]. J Mol Biol, 311(4), 735-749. doi: 10.1006/jmbi.2001.4876
Wu, P. Y., Lin, Y. C., Chang, C. L., Lu, H. T., Chin, C. H., Hsu, T. T., . . . Sun, S. H. (2009). Functional decreases in P2X7 receptors are associated with retinoic acid-induced neuronal differentiation of Neuro-2a neuroblastoma cells. [Research Support, Non-U.S. Gov't]. Cell Signal, 21(6), 881-891.
Yanagisawa, H., Komuta, Y., Kawano, H., Toyoda, M., & Sango, K. (2010). Pleiotrophin induces neurite outgrowth and up-regulates growth-associated protein (GAP)-43 mRNA through the ALK/GSK3beta/beta-catenin signaling in developing mouse neurons. [Research Support, Non-U.S. Gov't]. Neurosci Res, 66(1), 111-116. doi: 10.1016/j.neures.2009.10.002