請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/30805完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林淑華(Shu-Wha Lin) | |
| dc.contributor.author | Kang-Yi Su | en |
| dc.contributor.author | 蘇剛毅 | zh_TW |
| dc.date.accessioned | 2021-06-13T02:16:22Z | - |
| dc.date.available | 2009-02-27 | |
| dc.date.copyright | 2007-02-27 | |
| dc.date.issued | 2007 | |
| dc.date.submitted | 2007-02-13 | |
| dc.identifier.citation | Arimura N, Kaibuchi K (2005) Key regulators in neuronal polarity. Neuron 48:881-884.
Battye R, Stevens A, Jacobs JR (1999) Axon repulsion from the midline of the Drosophila CNS requires slit function. Development 126:2475-2481. Betz UA, Bloch W, van den Broek M, Yoshida K, Taga T, Kishimoto T, Addicks K, Rajewsky K, Muller W (1998) Postnatally induced inactivation of gp130 in mice results in neurological, cardiac, hematopoietic, immunological, hepatic, and pulmonary defects. J Exp Med 188:1955-1965. Bretin S, Reibel S, Charrier E, Maus-Moatti M, Auvergnon N, Thevenoux A, Glowinski J, Rogemond V, Premont J, Honnorat J, Gauchy C (2005) Differential expression of CRMP1, CRMP2A, CRMP2B, and CRMP5 in axons or dendrites of distinct neurons in the mouse brain. J Comp Neurol 486:1-17. Brion JP, Tremp G, Octave JN (1999) Transgenic expression of the shortest human tau affects its compartmentalization and its phosphorylation as in the pretangle stage of Alzheimer's disease. Am J Pathol 154:255-270. Brown M, Jacobs T, Eickholt B, Ferrari G, Teo M, Monfries C, Qi RZ, Leung T, Lim L, Hall C (2004) Alpha2-chimaerin, cyclin-dependent Kinase 5/p35, and its target collapsin response mediator protein-2 are essential components in semaphorin 3A-induced growth-cone collapse. J Neurosci 24:8994-9004. Byk T, Dobransky T, Cifuentes-Diaz C, Sobel A (1996) Identification and molecular characterization of Unc-33-like phosphoprotein (Ulip), a putative mammalian homolog of the axonal guidance-associated unc-33 gene product. J Neurosci 16:688-701. Chang YJ, Wu HL, Hamaguchi N, Hsu YC, Lin SW (2002) Identification of functionally important residues of the epidermal growth factor-2 domain of factor IX by alanine-scanning mutagenesis. Residues Asn(89)-Gly(93) are critical for binding factor VIIIa. J Biol Chem 277:25393-25399. Charrier E, Mosinger B, Meissirel C, Aguera M, Rogemond V, Reibel S, Salin P, Chounlamountri N, Perrot V, Belin MF, Goshima Y, Honnorat J, Thomasset N, Kolattukudy P (2006) Transient alterations in granule cell proliferation, apoptosis and migration in postnatal developing cerebellum of CRMP1-/- mice. Genes Cells 11:1337-1352. Cheng HJ, Nakamoto M, Bergemann AD, Flanagan JG (1995) Complementary gradients in expression and binding of ELF-1 and Mek4 in development of the topographic retinotectal projection map. Cell 82:371-381. Chi P, Greengard P, Ryan TA (2001) Synapsin dispersion and reclustering during synaptic activity. Nat Neurosci 4:1187-1193. Chilton JK (2006) Molecular mechanisms of axon guidance. Dev Biol 292:13-24. Cohen-Salmon M, Crozet F, Rebillard G, Petit C (1997) Cloning and characterization of the mouse collapsin response mediator protein-1, Crmp1. Mamm Genome 8:349-351. Cole AR, Causeret F, Yadirgi G, Hastie CJ, McLauchlan H, McManus EJ, Hernandez F, Eickholt BJ, Nikolic M, Sutherland C (2006) Distinct priming kinases contribute to differential regulation of collapsin response mediator proteins by glycogen synthase kinase-3 in vivo. J Biol Chem 281:16591-16598. D'Hooge R, De Deyn PP (2001) Applications of the Morris water maze in the study of learning and memory. Brain Res Brain Res Rev 36:60-90. De Camilli P, Cameron R, Greengard P (1983) Synapsin I (protein I), a nerve terminal-specific phosphoprotein. I. Its general distribution in synapses of the central and peripheral nervous system demonstrated by immunofluorescence in frozen and plastic sections. J Cell Biol 96:1337-1354. Deo RC, Schmidt EF, Elhabazi A, Togashi H, Burley SK, Strittmatter SM (2004) Structural bases for CRMP function in plexin-dependent semaphorin3A signaling. Embo J 23:9-22. Dickson BJ (2002) Molecular mechanisms of axon guidance. Science 298:1959-1964. Eddy NB, Leimbach D (1953) Synthetic analgesics. II. Dithienylbutenyl- and dithienylbutylamines. J Pharmacol Exp Ther 107:385-393. Fukada M, Watakabe I, Yuasa-Kawada J, Kawachi H, Kuroiwa A, Matsuda Y, Noda M (2000) Molecular characterization of CRMP5, a novel member of the collapsin response mediator protein family. J Biol Chem 275:37957-37965. Fukata Y, Itoh TJ, Kimura T, Menager C, Nishimura T, Shiromizu T, Watanabe H, Inagaki N, Iwamatsu A, Hotani H, Kaibuchi K (2002) CRMP-2 binds to tubulin heterodimers to promote microtubule assembly. Nat Cell Biol 4:583-591. Goshima Y, Nakamura F, Strittmatter P, Strittmatter SM (1995) Collapsin-induced growth cone collapse mediated by an intracellular protein related to UNC-33. Nature 376:509-514. Greengard P, Browning MD, McGuinness TL, Llinas R (1987) Synapsin I, a phosphoprotein associated with synaptic vesicles: possible role in regulation of neurotransmitter release. Adv Exp Med Biol 221:135-153. Grundke-Iqbal I, Iqbal K, Tung YC, Quinlan M, Wisniewski HM, Binder LI (1986) Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology. Proc Natl Acad Sci U S A 83:4913-4917. Gu Y, Ihara Y (2000) Evidence that collapsin response mediator protein-2 is involved in the dynamics of microtubules. J Biol Chem 275:17917-17920. Gu Y, Hamajima N, Ihara Y (2000) Neurofibrillary tangle-associated collapsin response mediator protein-2 (CRMP-2) is highly phosphorylated on Thr-509, Ser-518, and Ser-522. Biochemistry 39:4267-4275. Hedgecock EM, Culotti JG, Hall DH (1990) The unc-5, unc-6, and unc-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. elegans. Neuron 4:61-85. Henkemeyer M, Orioli D, Henderson JT, Saxton TM, Roder J, Pawson T, Klein R (1996) Nuk controls pathfinding of commissural axons in the mammalian central nervous system. Cell 86:35-46. Hollander MC, Philburn RT, Patterson AD, Velasco-Miguel S, Friedberg EC, Linnoila RI, Fornace AJ, Jr. (2005) Deletion of XPC leads to lung tumors in mice and is associated with early events in human lung carcinogenesis. Proc Natl Acad Sci U S A 102:13200-13205. Huber AB, Kolodkin AL, Ginty DD, Cloutier JF (2003) Signaling at the growth cone: ligand-receptor complexes and the control of axon growth and guidance. Annu Rev Neurosci 26:509-563. Inagaki N, Chihara K, Arimura N, Menager C, Kawano Y, Matsuo N, Nishimura T, Amano M, Kaibuchi K (2001) CRMP-2 induces axons in cultured hippocampal neurons. Nat Neurosci 4:781-782. Jassen AK, Yang H, Miller GM, Calder E, Madras BK (2006) Receptor regulation of gene expression of axon guidance molecules: implications for adaptation. Mol Pharmacol 70:71-77. Jin Z, Strittmatter SM (1997) Rac1 mediates collapsin-1-induced growth cone collapse. J Neurosci 17:6256-6263. Kalil K (1996) Growth cone behaviors during axon guidance in the developing cerebral cortex. Prog Brain Res 108:31-40. Keleman K, Dickson BJ (2001) Short- and long-range repulsion by the Drosophila Unc5 netrin receptor. Neuron 32:605-617. Kennedy MB (1997) The postsynaptic density at glutamatergic synapses. Trends Neurosci 20:264-268. Kennedy TE, Serafini T, de la Torre JR, Tessier-Lavigne M (1994) Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord. Cell 78:425-435. Kidd T, Bland KS, Goodman CS (1999) Slit is the midline repellent for the robo receptor in Drosophila. Cell 96:785-794. Kondo S, Okuda A, Sato H, Tachikawa N, Terashima M, Kanegae Y, Saito I (2003) Simultaneous on/off regulation of transgenes located on a mammalian chromosome with Cre-expressing adenovirus and a mutant loxP. Nucleic Acids Res 31:e76. Kosik KS, Joachim CL, Selkoe DJ (1986) Microtubule-associated protein tau (tau) is a major antigenic component of paired helical filaments in Alzheimer disease. Proc Natl Acad Sci U S A 83:4044-4048. Kullander K, Mather NK, Diella F, Dottori M, Boyd AW, Klein R (2001) Kinase-dependent and kinase-independent functions of EphA4 receptors in major axon tract formation in vivo. Neuron 29:73-84. Lee EC, Yu D, Martinez de Velasco J, Tessarollo L, Swing DA, Court DL, Jenkins NA, Copeland NG (2001) A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73:56-65. Leung T, Ng Y, Cheong A, Ng CH, Tan I, Hall C, Lim L (2002) p80 ROKalpha binding protein is a novel splice variant of CRMP-1 which associates with CRMP-2 and modulates RhoA-induced neuronal morphology. FEBS Lett 532:445-449. Lin CP, Wedeen VJ, Chen JH, Yao C, Tseng WY (2003) Validation of diffusion spectrum magnetic resonance imaging with manganese-enhanced rat optic tracts and ex vivo phantoms. Neuroimage 19:482-495. Liu BP, Strittmatter SM (2001) Semaphorin-mediated axonal guidance via Rho-related G proteins. Curr Opin Cell Biol 13:619-626. Lu Q, Wood JG (1993) Functional studies of Alzheimer's disease tau protein. J Neurosci 13:508-515. Luo Y, Raible D, Raper JA (1993) Collapsin: a protein in brain that induces the collapse and paralysis of neuronal growth cones. Cell 75:217-227. McIlvain V, McCasland JS (2006) GAP-43 heterozygous mice show delayed barrel patterning, differentiation of radial glia, and downregulation of GAP-43. Anat Rec A Discov Mol Cell Evol Biol 288:143-157. Minturn JE, Fryer HJ, Geschwind DH, Hockfield S (1995) TOAD-64, a gene expressed early in neuronal differentiation in the rat, is related to unc-33, a C. elegans gene involved in axon outgrowth. J Neurosci 15:6757-6766. Mitsui N, Inatome R, Takahashi S, Goshima Y, Yamamura H, Yanagi S (2002) Involvement of Fes/Fps tyrosine kinase in semaphorin3A signaling. Embo J 21:3274-3285. Morales M, Fifkova E (1989) Distribution of MAP2 in dendritic spines and its colocalization with actin. An immunogold electron-microscope study. Cell Tissue Res 256:447-456. Nakamura F, Kalb RG, Strittmatter SM (2000) Molecular basis of semaphorin-mediated axon guidance. J Neurobiol 44:219-229. Ozer RS, Halpain S (2000) Phosphorylation-dependent localization of microtubule-associated protein MAP2c to the actin cytoskeleton. Mol Biol Cell 11:3573-3587. Pfenninger KH, Laurino L, Peretti D, Wang X, Rosso S, Morfini G, Caceres A, Quiroga S (2003) Regulation of membrane expansion at the nerve growth cone. J Cell Sci 116:1209-1217. Quach TT, Duchemin AM, Rogemond V, Aguera M, Honnorat J, Belin MF, Kolattukudy PE (2004) Involvement of collapsin response mediator proteins in the neurite extension induced by neurotrophins in dorsal root ganglion neurons. Mol Cell Neurosci 25:433-443. Quach TT, Mosinger B, Jr., Ricard D, Copeland NG, Gilbert DJ, Jenkins NA, Stankoff B, Honnorat J, Belin MF, Kolattukudy P (2000) Collapsin response mediator protein-3/unc-33-like protein-4 gene: organization, chromosomal mapping and expression in the developing mouse brain. Gene 242:175-182. Quinn CC, Gray GE, Hockfield S (1999) A family of proteins implicated in axon guidance and outgrowth. J Neurobiol 41:158-164. Roberts LA, Large CH, Higgins MJ, Stone TW, O'Shaughnessy CT, Morris BJ (1998) Increased expression of dendritic mRNA following the induction of long-term potentiation. Brain Res Mol Brain Res 56:38-44. Schwarting GA, Kostek C, Ahmad N, Dibble C, Pays L, Puschel AW (2000) Semaphorin 3A is required for guidance of olfactory axons in mice. J Neurosci 20:7691-7697. Serafini T, Kennedy TE, Galko MJ, Mirzayan C, Jessell TM, Tessier-Lavigne M (1994) The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6. Cell 78:409-424. Shih JY, Yang SC, Hong TM, Yuan A, Chen JJ, Yu CJ, Chang YL, Lee YC, Peck K, Wu CW, Yang PC (2001) Collapsin response mediator protein-1 and the invasion and metastasis of cancer cells. J Natl Cancer Inst 93:1392-1400. Steeg PS (2001) Collapsin response mediator protein-1: a lung cancer invasion suppressor gene with nerve. J Natl Cancer Inst 93:1364-1365. Strittmatter SM, Fankhauser C, Huang PL, Mashimo H, Fishman MC (1995) Neuronal pathfinding is abnormal in mice lacking the neuronal growth cone protein GAP-43. Cell 80:445-452. Suzuki Y, Nakagomi S, Namikawa K, Kiryu-Seo S, Inagaki N, Kaibuchi K, Aizawa H, Kikuchi K, Kiyama H (2003) Collapsin response mediator protein-2 accelerates axon regeneration of nerve-injured motor neurons of rat. J Neurochem 86:1042-1050. Taniguchi M, Yuasa S, Fujisawa H, Naruse I, Saga S, Mishina M, Yagi T (1997) Disruption of semaphorin III/D gene causes severe abnormality in peripheral nerve projection. Neuron 19:519-530. Tessier-Lavigne M, Goodman CS (1996) The molecular biology of axon guidance. Science 274:1123-1133. Thiebot MH, Martin P, Puech AJ (1992) Animal behavioural studies in the evaluation of antidepressant drugs. Br J Psychiatry Suppl:44-50. Uchida Y, Ohshima T, Sasaki Y, Suzuki H, Yanai S, Yamashita N, Nakamura F, Takei K, Ihara Y, Mikoshiba K, Kolattukudy P, Honnorat J, Goshima Y (2005) Semaphorin3A signalling is mediated via sequential Cdk5 and GSK3beta phosphorylation of CRMP2: implication of common phosphorylating mechanism underlying axon guidance and Alzheimer's disease. Genes Cells 10:165-179. Vernino S, Tuite P, Adler CH, Meschia JF, Boeve BF, Boasberg P, Parisi JE, Lennon VA (2002) Paraneoplastic chorea associated with CRMP-5 neuronal antibody and lung carcinoma. Ann Neurol 51:625-630. Wang LH, Strittmatter SM (1996) A family of rat CRMP genes is differentially expressed in the nervous system. J Neurosci 16:6197-6207. Wang LH, Strittmatter SM (1997) Brain CRMP forms heterotetramers similar to liver dihydropyrimidinase. J Neurochem 69:2261-2269. Wettschureck N, Moers A, Hamalainen T, Lemberger T, Schutz G, Offermanns S (2004) Heterotrimeric G proteins of the Gq/11 family are crucial for the induction of maternal behavior in mice. Mol Cell Biol 24:8048-8054. Wilkinson DG (2001) Multiple roles of EPH receptors and ephrins in neural development. Nat Rev Neurosci 2:155-164. Yadav N, Lee J, Kim J, Shen J, Hu MC, Aldaz CM, Bedford MT (2003) Specific protein methylation defects and gene expression perturbations in coactivator-associated arginine methyltransferase 1-deficient mice. Proc Natl Acad Sci U S A 100:6464-6468. Yu HH, Kolodkin AL (1999) Semaphorin signaling: a little less per-plexin. Neuron 22:11-14. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/30805 | - |
| dc.description.abstract | 在神經發育的過程中，調控神經細胞軸突和樹突延展及潰縮的機制引導神經纖維精準達到標的組織或臟器關係到正常生理功能的運作。多年來已有許多的蛋白家族被發現與此機制有高度相關性，最具代表性即Semaphorin家族，其中又以Semaphorin 3A研究最為明確；經由細胞膜上Semaphorin 3A所傳導的訊息，可被膜內一群名為CRMP（Collapsin Response Mediator Protein）家族所承續。CRMP可以藉由和細胞骨架相關蛋白質或是和細胞骨架本體的交互聯結，促使骨架聚合化或是去聚合化達到神經纖維延展或潰縮。CRMP-1為CRMP蛋白家族的成員之一，過去研究顯示CRMP-1高度專一表現於神經系統中，尤其以大腦的海馬迴表現量最多，然而體外細胞培養的結果對於CRMP-1參與神經纖維延展或是潰縮的訊息傳遞仍有待釐。2001年發現CRMP-1的表現量和肺癌轉移和臨床癒後有負相關性，且定義為腫瘤轉移抑制基因。為研究CRMP-1在生物體內所扮演的角色及參與之功能，本論文以條件式基因剔除策略建立了CRMP-1基因缺乏小鼠；CRMP-1基因剔除小鼠可以正常生長及繁衍子代，外觀上與基礎血液生化檢驗亦和野生型小鼠無異，且不因老化而易產生自發性腫瘤。根據CRMP-1高表現的區域進一步分析，發現基因剔除小鼠的海馬迴及胼胝體雖然其結構和神經叢數量與野生型小鼠無異，但是經由MAP2（Microtubule-Associate Protein）螢光染色，發現在基因剔除小鼠CA1區域的樹突發育有潰縮及發育不整的表型，且和樹突相關的PSD95（Post-Synaptic Density）表現量也有明顯下降；此外，雖然CRMP-1基因剔除小鼠在軸突的發育以及構造上和野生型小鼠無明顯差異，然而和軸突以及神經電位可塑性有關的GAP-43（Growth-Associated Protein）在CA1區域表現量有顯著減少。這些表型也使得CRMP-1基因缺乏的小鼠在海馬迴CA1區域的長效性神經電位受到了抑制，相對影響到小鼠在水迷宮實驗中的空間學習和記憶能力。除此之外，在強制性游泳的實驗中，CRMP-1基因缺乏小鼠也表現出相對沮喪、停滯不動的情緒和行為反應，推測CRMP-1可能也參與了情緒調控的機制。綜言之，本論文成功以條件式基因剔除策略建立了CRMP-1基因缺乏小鼠，發現其樹突發育受到影響，使得與記憶相關的長效性神經電位受到抑制，造成空間學習與記憶能力缺損。另一方面，CRMP-1基因缺乏小鼠小腦發育也受到影響，除了重量較野生型小鼠輕外，小腦十個由顆粒層形成的摺葉中，編號第VIb的摺葉在發育上也有缺損，不明顯，推測CRMP-1亦調控小腦的生長與發育。 | zh_TW |
| dc.description.abstract | Brain function needs the precise anatomical and histological connections generated by exquisitely specific axonal guidance system during development. Neuronal outgrowth is directed by attracting by attracting and repelling signaling molecules. Well known guidance cues are semaphorins/collapsins. Semaphorin 3A, also known as Collapsin-1 is thought to be an initiator of signaling involved in neural growth cone outgrowth. Collapsin response mediator proteins (CRMPs) are a family of cytosolic phosphoproteins that mediate the signal from Semaphorin 3A. Collapsing response mediator protein-1 (CRMP-1) was initially identified in brain and has been implicated in plexin-dependent neuronal function. The high amino acid sequence identity among the five CRMPs has hindered determination of the functions of each individual CRMP. In 2001, CRMP-1 had also been characterized as a tumor metastasis suppressor gene dependent on its role in lung cancer metastasis and clinical outcome. In order to study the physiological function of CRMP-1 in vivo, we generated viable and fertile CRMP-1 knockout (CRMP-1-/-) mice with no evidence of gross abnormality in the major organs and difference in hematological and biochemical analysis between wild-type mice. According to high CRMP-1 expression in the hippocampus, we analyzed the formation of axon and dendrite there by staining with neural markers. CRMP-1-/- mice exhibited intense MAP2 staining in the proximal portion of the dendrites, but reduced and disorganized MAP2 staining in the distal dendrites of hippocampal CA1 pyramidal cells. Immunoreactivity to GAP-43 and PSD95 (a postsynaptic membrane adherent cytoskeletal protein) was also decreased in the CA1 region of the knockout mice. These changes were consistent with the mutant mice showing a reduction in long-term potentiation (LTP) in the CA1 region and impaired performance in hippocampal-dependent spatial learning and memory tests. CRMP-1-/- mice showed a normal synapsin I labeling pattern in CA1 and normal paired pulse facilitation. In addition, CRMP-1-/- also showed more depressed in forced swimming test suggested that CRMP-1 may involved in emotion control. These findings provide the first evidence suggesting that CRMP-1 may be involved in proper neurite outgrowth in the adult hippocampus and that loss of CRMP-1 may affect LTP maintenance and spatial learning and memory. Furthermore, CRMP-1-/- mice also exhibit more depressed in forced swimming test compared with wild-type mice suggested that CRMP-1 may regulate emotion response. On the other hand, CRMP-1-/- mice also show defect in cerebellum development. Mice deficient in CRMP-1 not only showed reduced weight in cerebellum but also lack the normal organic structure in VIb lobe suggested that CRMP-1 also involved in cerebellum development. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T02:16:22Z (GMT). No. of bitstreams: 1 ntu-96-D90448005-1.pdf: 16127262 bytes, checksum: bcd053d013747682460fb6a48816d33a (MD5) Previous issue date: 2007 | en |
| dc.description.tableofcontents | 口試委員會審定書………………………………………………………i
感言……………………………………………………………………ii 總目錄……………………………………………………………………1 圖目錄……………………………………………………………………5 表目錄……………………………………………………………………7 附錄目錄…………………………………………………………………8 縮寫表……………………………………………………………………9 中文摘要………………………………………………………………11 英文摘要………………………………………………………………13 第一章 導論…………………………………………………………15 1.1本章概要………………………………………………………… 15 1.2神經纖維在發育過程中的導向規則…………………………… 15 1.3調控神經纖維導向之蛋白質表現與作用……………………… 16 1.4 CRMP家族在神經纖維導向過程中的訊息傳遞…………………17 1.5 CRMP-1基因、蛋白質與功能性研究簡介………………………19 1.6本論文研究動機及實驗策略…………………………………… 21 1.7本論文研究成果概述…………………………………………… 22 第二章 實驗材料與方法………………………………………… 25 2.1 本章概要…………………………………………………………25 2.2 CRMP-1基因剔除質體構築………………………………………25 2.2.1質體限制酵素切割、部份切割、片段純化、電泳分析和接 合………………………………………………………………… 25 2.2.2質體轉型勝任細胞、大量複製、純化……………………… 26 2.2.3 CRMP-1基因剔除質體構築流程………………………………26 2.2.4 CRMP-1基因剔除再篩選質體構築流程………………………28 2.3胚胎幹細胞培養、轉染和篩選………………………………… 28 2.3.1基層纖維母細胞製備和mitomycin C處理……………………28 2.3.2胚胎幹細胞培養……………………………………………… 29 2.3.3質體轉染胚胎幹細胞、正負篩選…………………………… 30 2.3.4胚胎幹細胞DNA萃取……………………………………………31 2.4 嵌合鼠製備、育種和照護………………………………………31 2.4.1囊胚細胞顯微注射、子宮植入……………………………… 31 2.4.2嵌合鼠育種和照護…………………………………………… 32 2.5 CRMP-1基因剔除小鼠基因型、mRNA及蛋白質表現分析和 定量…………………………………………………………………33 2.5.1小鼠尾部DNA萃取………………………………………………33 2.5.2南方點墨法分析……………………………………………… 33 2.5.3聚合脢聯鎖反應（PCR）基因型鑑定…………………………35 2.5.4小鼠腦部RNA萃取………………………………………………35 2.5.5北方點墨法分析……………………………………………… 36 2.5.6反轉錄反應…………………………………………………… 37 2.5.7小鼠腦部分區蛋白質萃取…………………………………… 37 2.5.8西方點墨法分析……………………………………………… 38 2.5.9蛋白質表現半定量分析……………………………………… 38 2.6小鼠基礎血液學、生化學篩檢分析…………………………… 39 2.6.1小鼠眼角採血………………………………………………… 39 2.6.2基礎血液及生化學篩檢……………………………………… 39 2.7小鼠組織病理檢體取得、處理和製片………………………… 40 2.7.1小鼠腦部灌流………………………………………………… 40 2.7.2腦部檢體處理、固定和切片………………………………… 40 2.7.3小鼠脊髓及背角神經結分離………………………………… 41 2.8組織病理染色分析……………………………………………… 42 2.8.1切片脫臘、抗原再現………………………………………… 42 2.8.2甲酚紫（cresyl violet）染色 (Nissl staining)……… 42 2.8.3 DiI神經纖維投射染色……………………………………… 43 2.8.4蘇木紫和伊紅染色…………………………………………… 43 2.8.5組織免疫螢光染色…………………………………………… 44 2.9大腦電生理長效性電位分析…………………………………… 45 2.10小鼠動物行為實驗分析…………………………………………46 2.10.1水迷宮學習記憶實驗…………………………………………46 2.10.2小鼠行動能力鑑定……………………………………………47 2.10.3旋轉滾筒平衡實驗……………………………………………48 2.10.4痛覺耐受測試實驗………………………………………… 48 第三章 實驗結果……………………………………………………51 3.1 本章概要…………………………………………………………51 3.2南方點墨法確認質體於胚胎幹細胞中正確同質互換………… 51 3.2.1第一次同質互換標的篩選…………………………………… 51 3.2.2第二次同質互換標的再篩選………………………………… 52 3.3嵌合鼠出生及回復交配至C57BL/6基因背景……………………53 3.4確認CRMP-1基因、mRNA和蛋白質在剔除小鼠中無表現……… 53 3.4.1確認CRMP-1基因野生、單套剔除、雙套剔除和條件式 基剔除等基因型.……………………………………………53 3.4.2反轉錄聚合脢聯鎖反應發現CRMP-1基因剔除小鼠可轉 錄出較短不全的mRNA………………………………………… 54 3.4.3 北方點墨法確認CRMP-1基因剔除小鼠無mRNA轉錄…………55 3.4.4西方點墨法確認CRMP-1基因剔除小鼠無CRMP-1蛋白 質表現………………………………………………………… 55 3.4.5 CRMP-1蛋白質在腦中以海馬迴表現量最多…………………56 3.5 CRMP-1基因剔除小鼠可以正常生長且繁殖後代，外觀與野生 型小鼠無異………………………………………………………57 3.6 CRMP-1基因剔除小鼠在海馬迴結構上正常，但樹突發育和延 展有明顯缺損……………………………………………………58 3.6.1海馬海馬迴結構及胼胝體神經叢數目與野生型小鼠無異… 58 3.6.2 CRMP-1基因剔除小鼠於海馬迴CA1區域之樹突分佈異常… 58 3.6.3突觸後密度蛋白質PSD95表現量下降…………………………59 3.6.4與生長相關之GAP-43蛋白質表現量下降…………………… 60 3.6.5在軸突的形成與結構上野生型小鼠和基因剔除小鼠異…… 61 3.6.6 CRMP-1基因剔除小鼠在脊髓及週邊神經上無明顯異常……62 3.7 CRMP-1基因剔除小鼠無法維持電擊刺激下所誘發的長效性電位…………………………………………………………………………62 3.8 CRMP-1基因剔除小鼠在空間學習和記憶能力上有明顯缺損…63 3.9 CRMP-1基因剔除小鼠在平衡、痛覺感受和其它行為測試實驗 中和野生型小鼠無異……………………………………………64 3.10 CRMP-1基因剔除小鼠除了海馬迴病變外，小腦的發育也有 明顯異常且在情緒反應實驗中亦顯得較易沮喪………………65 3.10.1本節概述………………………………………………………65 3.10.2 CRMP-1基因剔除小鼠小腦重量較輕且發育異…………… 65 3.10.3 CRMP-1基因剔除小鼠在穿縮逃脫電擊反應測試中有遲緩 傾向………………………………………………………… 66 3.10.4 CRMP-1基因剔除小鼠在情緒測試中較易有沮喪的反…… 66 第四章 實驗討論……………………………………………………69 4.1 本章概要…………………………………………………………69 4.2利用基因剔除技術建立CRMP-1基因缺乏小鼠………………… 69 4.3 CRMP-1基因剔除小鼠轉錄出短小片段之mRNA…………………71 4.4 CRMP-1基因剔除造成海馬迴CA1樹突異常…………………… 72 4.5基因剔除小鼠軸突形成與發育………………………………… 73 4.6 CRMP-1蛋白質與樹突分佈和發育………………………………74 4.7 CRMP-1與神經可塑性……………………………………………74 4.8 CRMP-1與小鼠空間學習能力及行為反應………………………75 4.9 CRMP-1和腫瘤形成和轉移………………………………………76 4.10 CRMP-1和小腦發育及功能…………………………………… 76 第五章 結論與展望…………………………………………………79 參考文獻………………………………………………………………83 圖………………………………………………………………………97 表…………………………………………………………………… 137 附錄………………………………………………………………… 141 已發表之論文……………………………………………………… 146 | |
| dc.language.iso | zh-TW | |
| dc.subject | 基因剔除 | zh_TW |
| dc.subject | CRMP-1 | zh_TW |
| dc.subject | 空間學習記憶 | zh_TW |
| dc.subject | 長效性電位 | zh_TW |
| dc.subject | 軸突形成 | zh_TW |
| dc.subject | 海馬迴 | zh_TW |
| dc.subject | learning and memory | en |
| dc.subject | CRMP-1 | en |
| dc.subject | gene targeting | en |
| dc.subject | cre-loxP | en |
| dc.subject | hippocampus | en |
| dc.subject | axon guidance | en |
| dc.subject | apical dendrite formation | en |
| dc.subject | long-term potentiation | en |
| dc.subject | Morris Water Maze | en |
| dc.title | 以建立CRMP-1基因剔除小鼠動物模型研究其在腦部生理功能的角色 | zh_TW |
| dc.title | Gene Targeting of CRMP-1 in Mice as a Model For Brain Functional Study | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 95-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 楊泮池(Pan-Chyr Yang),符文美(Wen-Mei Fu),謝松蒼(Sung-Tsang Hsieh),陳志宏(Jyh-Horng Chen),李芳仁(Fang-Jen Lee),陳志成(Chih-Cheng Chen),杜邦憲(Pang-Hsien Tu) | |
| dc.subject.keyword | CRMP-1,基因剔除,海馬迴,軸突形成,長效性電位,空間學習記憶, | zh_TW |
| dc.subject.keyword | CRMP-1,gene targeting,cre-loxP,hippocampus,axon guidance,apical dendrite formation,long-term potentiation,Morris Water Maze,learning and memory, | en |
| dc.relation.page | 145 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2007-02-13 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 分子醫學研究所 | zh_TW |
| 顯示於系所單位： | 分子醫學研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-96-1.pdf 未授權公開取用 | 15.75 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。