簡易檢索 / 詳目顯示

回結果列表
研究生： 周瑀涵
Chou, Yu-Han
論文名稱： 訊息蛋白SH2B1β增進PC12細胞之神經修復能力
The Adaptor Protein SH2B1β Enhances Neuronal Regeneration in PC12 Cells
指導教授： 陳令儀
Chen, Linyi
口試委員:
學位類別： 碩士
Master
系所名稱： 生命科學暨醫學院 - 分子醫學研究所
Institute of Molecular Medicine
論文出版年： 2009
畢業學年度： 97
語文別： 中文
論文頁數： 80
中文關鍵詞： SH2B1β 、神經修復
外文關鍵詞： SH2B1β, neuronal regeneration
相關次數： 點閱：2  下載：0 
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報

    • 在神經系統發育的過程，神經細胞為了存活而相互競爭有限的營養因子。神經細胞若無法得到足夠的營養因子，就會開始走向細胞凋亡。雖然細胞凋亡在神經系統發育是必然的過程，但是成熟的神經細胞不正常的死亡而使結構及功能日益失去就會造成神經退化的疾病，如阿茲海默症與帕金森氏症。SH2B1β是一種訊息接合蛋白，參與許多生長因子的訊息傳遞路徑，像是神經生長因子(nerve growth factor, NGF)及纖維母細胞生長因子一(fibroblast growth factor 1, FGF1)下游的訊息傳遞。NGF可誘導大鼠腎上腺髓質嗜鉻細胞瘤細胞株 (rat pheochromocytoma cell line, 以下簡稱PC12 細胞)的神經分化，進而分化成為類交感神經細胞。先前的研究發現，SH2B1β可以增加PC12細胞由NGF及FGF1所誘導的神經分化，此外，我們最近的研究發現，SH2B1β還可以減少由氧化壓力所造成的細胞死亡，而氧化壓力也被認為是造成神經退化疾病原因之一。因此，我們推測SH2B1β對於神經退化及神經修復可能扮演一些重要的角色。我們發現SH2B1β可以減少由缺乏NGF所造成的神經退化，也可以增加軸突受傷之後的修復，因此我們更進一步探討SH2B1β如何增進神經細胞受傷之後的修復。我們發現SH2B1β可以透過許多機制來增加神經修復。一個被認為是神經修復標定的蛋白質--- GAP-43，在大量表現SH2B1β的細胞中高度表現，因此SH2B1β可能可以調控GAP-43表現而增加PC12細胞的修復能力。另外，STAT3、NCAM-L1以及N-cad的表現量隨著修復時間的增加，都有逐漸下降的趨勢，並且它們的表現量在大量表現SH2B1β的細胞中都較少。NCAM-L1及N-cadherin這些蛋白質可使細胞移動的速度增加，可以更快速的到傷口區域來促進修復。

    During development of vertebrate nervous system, neurons compete for limited target-derived trophic factors for survival. Insufficient neurotrophic support renders cells undergoing programmed cell death. Although cell death is a normal process during neuronal development, abnormal cell death of matured neurons and progressive loss of structure or function results in neurodegeneration such as Alzheimer’s disease and Parkinson’s disease. The adaptor protein SH2B1β is a signaling molecule that participates in various signaling pathway including nerve growth factor (NGF) and fibroblast growth factor (FGF) signaling. SH2B1β also enhances NGF- as well as FGF1-induced neurite outgrowth in PC12 cell, a well-established neuronal model that can be differentiated into sympathetic-like neurons upon NGF challenge. Moreover, our recent results suggest that SH2B1β could reduce oxidative stress-induced cell death. Oxidative stress has been linked to the neuronal cell death, which is associated with various neurodegenerative conditions. These results lead us to hypothesize that SH2B1β may play a role in neuronal degeneration and/or regeneration. In this study, I have shown that SH2B1β reduces neuronal degeneration during NGF deprivation and enhances regeneration after nerve injury. In addition, SH2B1β regulates the pro-inflammatory cytokines-induced signaling. To understand the mechanisms by which SH2B1β exerts its effect, we use wound healing assay as an injury model to investigate how SH2B1□ enhances neuronal regeneration. I have found that SH2B1□-overexpressing cells express higher level of growth-associated protein-43 (GAP-43), that may promote regeneration. In addition, down-regulation of STAT3, NCAM-L1, and N-cadherin might affect cell motility which leads cells migrate to the wounded area to promote regeneration. Taken together, SH2B1β may utilize multiple mechanisms to promote regeneration.

    誌謝 Abstract 中文摘要 Index Abbreviations Introduction Fig. I Neuronal survival pathways induced by the binding of NGF to its receptor TrkA Fig. II Schematics of SH2B family members Material and Methods Reagents Cell Culture NGF deprivation Wound healing assay Tracking analysis Injury of differentiated PC12 cells Immunoblotting Western blotting using Infrared imaging system (Odyssey detection) Total RNA preparation Reverse transcription and semi-quantitative real-time polymerase chain reaction BrdU incorporation staining Statistical analysis Results SH2B1β reduces neuronal degeneration during NGF deprivation SH2B1β enhances neurite regrowth after injury SH2B1β enhances regeneration after wounding SH2B1β modulates cytokines secretion during wound healing process The level of STAT3 is decreased during neuronal regeneration High expression of GAP-43 in SH2B1β-overexpressing cells SH2B1β may enhance regeneration through promoting cell migration Decreased expression of NCAM-L1 and N-cad is associated with neuronal regeneration SH2B1β does not induce cell proliferation to promote regeneration Discussion Reference Figures Fig. 1 Overexpressing SH2B1β reduces neuronal degeneration during NGF deprivation 40 Fig. 2 SH2B1β enhances neurite regrowth after injury Fig. 3 The expression of STAT3 is reduced during regeneration Fig. 4 Overexpressing SH2B1β enhances healing after mechanical injury Fig. 5 Overexpressing SH2B1β enhances cytokine-induced signaling Fig. 6 The mRNA level of cytokines is induced in response to wounding Fig. 7 Down-regulation of STAT3 expression during regeneration Fig. 8 The expression of GAP-43 in SH2B1β-overexpressing cells Fig. 9 PKC activator enhances cell migration Fig. 10 SH2B1β-overexpressing cells increase cell motility during healing Fig. 11 PKC activator enhances cell migration Fig. 12 Decreased expression of adhesion molecules during regeneration Fig. 13 Detection of cell proliferation using BrdU incorporation Fig. 14 Working model Tables Table 1. Reverse transcription protocol used in this thesis Table 2. Reaction temperature and time of reverse transcription Table 3. Sequences of the Q-PCR primers used in this thesis Table 4. Reaction temperature and time of Q-PCR Appendix Fig. A1 Serum concentration affects regeneration rate in PC12-GFP cells Fig. A2 High mRNA level of MMP-3 and MMP-10 in SH2B1β-overexpressing cells Fig. A3 Phosphorylation of CREB and ATF1 is increased after wounding

    1.Yuan, J., and Yankner, B. A. (2000) Nature 407, 802-809
    2.Hamburger, V., and Levi-Montalcini, R. (1949) J Exp Zool 111, 457-501
    3.Northcutt, R. G. (1989) Science 244, 993
    4.Korsching, S. (1993) J Neurosci 13, 2739-2748
    5.Raff, M. C., Barres, B. A., Burne, J. F., Coles, H. S., Ishizaki, Y., and Jacobson, M. D. (1993) Science 262, 695-700
    6.Klein, R., Jing, S. Q., Nanduri, V., O'Rourke, E., and Barbacid, M. (1991) Cell 65, 189-197
    7.Kaplan, D. R., Martin-Zanca, D., and Parada, L. F. (1991) Nature 350, 158-160
    8.Kaplan, D. R., Hempstead, B. L., Martin-Zanca, D., Chao, M. V., and Parada, L. F. (1991) Science 252, 554-558
    9.Fruman, D. A., Meyers, R. E., and Cantley, L. C. (1998) Annu Rev Biochem 67, 481-507
    10.Datta, S. R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., and Greenberg, M. E. (1997) Cell 91, 231-241
    11.Essers, M. A., Weijzen, S., de Vries-Smits, A. M., Saarloos, I., de Ruiter, N. D., Bos, J. L., and Burgering, B. M. (2004) EMBO J 23, 4802-4812
    12.Asada, S., Daitoku, H., Matsuzaki, H., Saito, T., Sudo, T., Mukai, H., Iwashita, S., Kako, K., Kishi, T., Kasuya, Y., and Fukamizu, A. (2007) Cell Signal 19, 519-527
    13.Du, K., and Montminy, M. (1998) J Biol Chem 273, 32377-32379
    14.Kane, L. P., Shapiro, V. S., Stokoe, D., and Weiss, A. (1999) Curr Biol 9, 601-604
    15.Riccio, A., Ahn, S., Davenport, C. M., Blendy, J. A., and Ginty, D. D. (1999) Science 286, 2358-2361
    16.Bonni, A., Brunet, A., West, A. E., Datta, S. R., Takasu, M. A., and Greenberg, M. E. (1999) Science 286, 1358-1362
    17.Friedlander, R. M. (2003) N Engl J Med 348, 1365-1375
    18.Allan, S. M., and Rothwell, N. J. (2001) Nat Rev Neurosci 2, 734-744
    19.Bossy-Wetzel, E., Schwarzenbacher, R., and Lipton, S. A. (2004) Nat Med 10 Suppl, S2-9
    20.Griffin, J. W., George, E. B., and Chaudhry, V. (1996) Baillieres Clin Neurol 5, 65-75
    21.Raff, M. C., Whitmore, A. V., and Finn, J. T. (2002) Science 296, 868-871
    22.Coyle, J. T., and Puttfarcken, P. (1993) Science 262, 689-695
    23.Klein, J. A., and Ackerman, S. L. (2003) J Clin Invest 111, 785-793
    24.Andersen, J. K. (2004) Nat Med 10 Suppl, S18-25
    25.Ischiropoulos, H., and Beckman, J. S. (2003) J Clin Invest 111, 163-169
    26.Barnham, K. J., Masters, C. L., and Bush, A. I. (2004) Nat Rev Drug Discov 3, 205-214
    27.Kim, B. Y., Han, M. J., and Chung, A. S. (2001) Free Radic Biol Med 30, 686-698
    28.Day, R. M., and Suzuki, Y. J. (2005) Dose Response 3, 425-442
    29.Davies, K. J. (1999) IUBMB Life 48, 41-47
    30.Rothwell, N. J., and Strijbos, P. J. (1995) Int J Dev Neurosci 13, 179-185
    31.Goldberg, J. L., and Barres, B. A. (2000) Annu Rev Neurosci 23, 579-612
    32.Makwana, M., and Raivich, G. (2005) FEBS J 272, 2628-2638
    33.Richardson, P. M., McGuinness, U. M., and Aguayo, A. J. (1980) Nature 284, 264-265
    34.Horner, P. J., and Gage, F. H. (2000) Nature 407, 963-970
    35.Snider, W. D., Zhou, F. Q., Zhong, J., and Markus, A. (2002) Neuron 35, 13-16
    36.Chen, Z. L., Yu, W. M., and Strickland, S. (2007) Annu Rev Neurosci 30, 209-233
    37.Qiu, J., Cai, D., and Filbin, M. T. (2002) Prog Brain Res 137, 381-387
    38.Qiu, J., Cai, D., Dai, H., McAtee, M., Hoffman, P. N., Bregman, B. S., and Filbin, M. T. (2002) Neuron 34, 895-903
    39.Neumann, S., Bradke, F., Tessier-Lavigne, M., and Basbaum, A. I. (2002) Neuron 34, 885-893
    40.Cai, D., Deng, K., Mellado, W., Lee, J., Ratan, R. R., and Filbin, M. T. (2002) Neuron 35, 711-719
    41.Gao, Y., Deng, K., Hou, J., Bryson, J. B., Barco, A., Nikulina, E., Spencer, T., Mellado, W., Kandel, E. R., and Filbin, M. T. (2004) Neuron 44, 609-621
    42.Schweizer, U., Gunnersen, J., Karch, C., Wiese, S., Holtmann, B., Takeda, K., Akira, S., and Sendtner, M. (2002) J Cell Biol 156, 287-297
    43.Shuai, K., and Liu, B. (2003) Nat Rev Immunol 3, 900-911
    44.Raivich, G., Bohatschek, M., Da Costa, C., Iwata, O., Galiano, M., Hristova, M., Nateri, A. S., Makwana, M., Riera-Sans, L., Wolfer, D. P., Lipp, H. P., Aguzzi, A., Wagner, E. F., and Behrens, A. (2004) Neuron 43, 57-67
    45.Qiu, J., Cafferty, W. B., McMahon, S. B., and Thompson, S. W. (2005) J Neurosci 25, 1645-1653
    46.Estus, S., Zaks, W. J., Freeman, R. S., Gruda, M., Bravo, R., and Johnson, E. M., Jr. (1994) J Cell Biol 127, 1717-1727
    47.Zhou, F. Q., Walzer, M. A., and Snider, W. D. (2004) Neuron 43, 1-2
    48.Cafferty, W. B., Gardiner, N. J., Das, P., Qiu, J., McMahon, S. B., and Thompson, S. W. (2004) J Neurosci 24, 4432-4443
    49.Regis, G., Pensa, S., Boselli, D., Novelli, F., and Poli, V. (2008) Semin Cell Dev Biol 19, 351-359
    50.Perlson, E., Hanz, S., Ben-Yaakov, K., Segal-Ruder, Y., Seger, R., and Fainzilber, M. (2005) Neuron 45, 715-726
    51.Hanz, S., Perlson, E., Willis, D., Zheng, J. Q., Massarwa, R., Huerta, J. J., Koltzenburg, M., Kohler, M., van-Minnen, J., Twiss, J. L., and Fainzilber, M. (2003) Neuron 40, 1095-1104
    52.Cavalli, V., Kujala, P., Klumperman, J., and Goldstein, L. S. (2005) J Cell Biol 168, 775-787
    53.Lindwall, C., and Kanje, M. (2005) Mol Cell Neurosci 29, 269-282
    54.Waetzig, V., and Herdegen, T. (2005) Mol Cell Neurosci 30, 67-78
    55.Abe, N., and Cavalli, V. (2008) Curr Opin Neurobiol 18, 276-283
    56.Yoshimura, T., Kawano, Y., Arimura, N., Kawabata, S., Kikuchi, A., and Kaibuchi, K. (2005) Cell 120, 137-149
    57.Zhou, F. Q., Walzer, M., Wu, Y. H., Zhou, J., Dedhar, S., and Snider, W. D. (2006) J Cell Sci 119, 2787-2796
    58.Zhou, F. Q., Zhou, J., Dedhar, S., Wu, Y. H., and Snider, W. D. (2004) Neuron 42, 897-912
    59.Hoke, A., Cheng, C., and Zochodne, D. W. (2000) Neuroreport 11, 1651-1654
    60.Grothe, C., and Nikkhah, G. (2001) Anat Embryol (Berl) 204, 171-177
    61.Jungnickel, J., Haase, K., Konitzer, J., Timmer, M., and Grothe, C. (2006) J Neurobiol 66, 940-948
    62.Cafferty, W. B., Gardiner, N. J., Gavazzi, I., Powell, J., McMahon, S. B., Heath, J. K., Munson, J., Cohen, J., and Thompson, S. W. (2001) J Neurosci 21, 7161-7170
    63.Kiryushko, D., Berezin, V., and Bock, E. (2004) Ann N Y Acad Sci 1014, 140-154
    64.Walsh, F. S., and Doherty, P. (1996) Curr Opin Cell Biol 8, 707-713
    65.Dihne, M., Bernreuther, C., Sibbe, M., Paulus, W., and Schachner, M. (2003) J Neurosci 23, 6638-6650
    66.Perrone-Bizzozero, N. I., Cansino, V. V., and Kohn, D. T. (1993) J Cell Biol 120, 1263-1270
    67.Benowitz, L. I., and Routtenberg, A. (1997) Trends Neurosci 20, 84-91
    68.Yankner, B. A., Benowitz, L. I., Villa-Komaroff, L., and Neve, R. L. (1990) Brain Res Mol Brain Res 7, 39-44
    69.Saika, T., Kiyama, H., Tohyama, M., and Matsunaga, T. (1993) Acta Otolaryngol Suppl 501, 80-84
    70.Tedeschi, A., Nguyen, T., Puttagunta, R., Gaub, P., and Di Giovanni, S. (2009) Cell Death Differ 16, 543-554
    71.Bomze, H. M., Bulsara, K. R., Iskandar, B. J., Caroni, P., and Skene, J. H. (2001) Nat Neurosci 4, 38-43
    72.Maures, T. J., Kurzer, J. H., and Carter-Su, C. (2007) Trends Endocrinol Metab 18, 38-45
    73.Greene, L. A. (1978) J Cell Biol 78, 747-755
    74.Riedel, H., Wang, J., Hansen, H., and Yousaf, N. (1997) J Biochem 122, 1105-1113
    75.Nelms, K., O'Neill, T. J., Li, S., Hubbard, S. R., Gustafson, T. A., and Paul, W. E. (1999) Mamm Genome 10, 1160-1167
    76.Wang, J., and Riedel, H. (1998) J Biol Chem 273, 3136-3139
    77.Qian, X., Riccio, A., Zhang, Y., and Ginty, D. D. (1998) Neuron 21, 1017-1029
    78.Rui, L., Herrington, J., and Carter-Su, C. (1999) J Biol Chem 274, 10590-10594
    79.Zhang, Y., Zhu, W., Wang, Y. G., Liu, X. J., Jiao, L., Liu, X., Zhang, Z. H., Lu, C. L., and He, C. (2006) J Cell Sci 119, 1666-1676
    80.Rui, L., and Carter-Su, C. (1998) J Biol Chem 273, 21239-21245
    81.Lin, W. F., Chen, C. J., Chang, Y. J., Chen, S. L., Chiu, I. M., and Chen, L. (2009) Cell Signal 21, 1060-1072
    82.Rui, L., Mathews, L. S., Hotta, K., Gustafson, T. A., and Carter-Su, C. (1997) Mol Cell Biol 17, 6633-6644
    83.O'Brien, K. B., O'Shea, J. J., and Carter-Su, C. (2002) J Biol Chem 277, 8673-8681
    84.Nishi, M., Werner, E. D., Oh, B. C., Frantz, J. D., Dhe-Paganon, S., Hansen, L., Lee, J., and Shoelson, S. E. (2005) Mol Cell Biol 25, 2607-2621
    85.Rui, L., and Carter-Su, C. (1999) Proc Natl Acad Sci U S A 96, 7172-7177
    86.Chen, L., and Carter-Su, C. (2004) Mol Cell Biol 24, 3633-3647
    87.Chen, L., Maures, T. J., Jin, H., Huo, J. S., Rabbani, S. A., Schwartz, J., and Carter-Su, C. (2008) Mol Endocrinol 22, 454-476
    88.Maures, T. J., Chen, L., and Carter-Su, C. (2009) Mol Endocrinol 23, 1077-1091
    89.Wang, X., Chen, L., Maures, T. J., Herrington, J., and Carter-Su, C. (2004) J Biol Chem 279, 133-141
    90.Sharma, G. D., He, J., and Bazan, H. E. (2003) J Biol Chem 278, 21989-21997
    91.Yeow, K., Cabane, C., Turchi, L., Ponzio, G., and Derijard, B. (2002) Biochem Biophys Res Commun 293, 112-119
    92.Weber, J. R., and Skene, J. H. (1998) J Neurosci 18, 5264-5274
    93.Di Giovanni, S., Knights, C. D., Rao, M., Yakovlev, A., Beers, J., Catania, J., Avantaggiati, M. L., and Faden, A. I. (2006) EMBO J 25, 4084-4096
    94.Dauer, D. J., Ferraro, B., Song, L., Yu, B., Mora, L., Buettner, R., Enkemann, S., Jove, R., and Haura, E. B. (2005) Oncogene 24, 3397-3408
    95.Kubota, K., Inoue, K., Hashimoto, R., Kumamoto, N., Kosuga, A., Tatsumi, M., Kamijima, K., Kunugi, H., Iwata, N., Ozaki, N., Takeda, M., and Tohyama, M. (2009) J Neurochem 110, 496-508
    96.Dawson, T. M., and Ginty, D. D. (2002) Nat Med 8, 450-451
    97.Lonze, B. E., and Ginty, D. D. (2002) Neuron 35, 605-623

    無法下載圖示 全文公開日期 本全文未授權公開 (校內網路)
    全文公開日期 本全文未授權公開 (校外網路)
    全文公開日期 本全文未授權公開 (國家圖書館：臺灣博碩士論文系統)
    QR CODE