臺灣博碩士論文加值系統

哺乳類動物的神經系統研究中發現，當神經發生損傷時，其周邊神經系統具有再生的能力，而在成熟的中樞神經系統損傷時卻缺乏了修復及再生的功能。近年來的研究指出，在中樞神經系統中存在了一些抑制物質（例如 Nogo-A蛋白和 proteoglycans等）會影響神經的生長及再生，這些抑制性物質目前已經被鑑定出來，它們是一群存在於中樞神經髓鞘的蛋白質：Nogo-A、MAG以及OMgp，而Nogo-A則是其中最重要的抑制蛋白。在臨床上對於中樞神經系統損傷的患者通常會立即給予高劑量人工合成的糖質皮質固醇（methylprednisolone；MP) 來治療，對於神經復原具有一定程度的療效。因此MP透過何種途徑，來達到治療效果是一個值得探討的研究主題，由於目前的研究指出影響神經再生的抑制蛋白主要是 Nogo-A；MP 作用是否透過影響Nogo-A上游啟動子的調節，進而影響其基因的表現，而對神經損傷有良好的復原效果為本論文主要探討的主題。
本實驗利用大白鼠的星狀細胞和寡突膠細胞為離體實驗系統，給予MP（1μM）處理後再分析 2.4kb、1.2kb以及 2.1kb等不同長度 Nogo-A 啟動子的表現。結果顯示當MP處理18小時後會影響Nogo-A 2.4kb的啟動子片段活性，使報導基因表現下降；而deletion 400bp的rNogo-A 2.1kb啟動子片段，則沒有前述的影響。當利用AMPA處理上述細胞，使產生興奮性毒性物質來模擬神經細胞受到傷害時的狀態，結果也發現 Nogo-A表現量並未如預期顯著增加；反而在AMPA 投予之下發現Nogo-A基因表現有下降的現象（在寡突膠細胞中最為明顯）。然而給予AMPA (星狀細胞200μM；寡突膠細胞100μM) 後再合併給予MP處理，可發現Nogo-A基因表現有更加明顯的抑制現象。
而在脊髓損傷的動物模式中也有同樣的情形；Nogo-A基因在損傷部位並沒有顯著的增加，反而在損傷部位鄰近組織發現Nogo-A的大量表現，而當予以MP治療後可觀察到Nogo-A表現受到抑制而表現量下降。在活體以及離體的實驗皆可證明MP對Nogo-A所具有的影響。另外從斑馬魚的動物實驗系統也觀察到，當同時給予MP 和AMPA處理的魚，其神經生長相較於對照組以及其他單獨處理藥物的組別有明顯神經樹突增加的現象；此結果顯示出MP 和AMPA同時處理，可能具有促進神經生長的協同作用存在。
綜合本實驗結果可知，給予MP處理對於Nogo-A基因表現確實具有抑制的作用，而且deletion 400bp的這段啟動子序列中可能含有一些重要的轉錄因子調控著MP對Nogo-A的影響。此外，在MP與AMPA的協同作用之下確實具有助於降低Nogo-A的表現並且促進神經生長的結果，然而其中機轉為何，值得未來更進一步的探討。

In mammalian nervous system, axons in the peripheral nervous system (PNS) can be regenerated after injury, whereas axons in the central nervous system (CNS) can not. The lack of ability of mammalian CNS axons to regenerate after spinal cord injury (SCI) maybe due to the glial scar formation will result in the generation of a number of proteins (e.g., Nogo proteins) and proteoglycans (e.g., chondroitin sulfate proteoglycans) then inhibit neurite outgrowth. Several of these inhibitory proteins are associated with CNS myelin have been characterized, including Nogo, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp), Nogo-A is the most important one that interferes with axon regrowth in the mammalian CNS and stimulates growth cone collapse in vitro. Methylpredinisolone (MP), a synthetic glucocorticoid (GC), is the known as the major therapeutic agent for acute spinal cord injury (SCI). MP, like other glucocorticoids, has broad effects on transcription factors including influencing cell viability.
In this study, we investigated the regulation of the upstream element of Nogo-A gene promoter to understanding essential mechanism of MP mediated of Nogo-A gene expression after CNS injury. We have established rat Nogo-A 2.4 kb, 1.2 kb and 2.1 kb promoter sequences-driven reporter genes for investigating the transcriptional activation process of the Nogo-A expression. The pGL3-Nogo-A- 2.4 and pGL3-Nogo-A-1.2 constructs was delivered into astrocytes or oligodendrocytes to investigate the possible mechanism of MP mediated Nogo-A gene expression.
The result shows that MP (1μM) down-regulates Nogo-A promoter activity but the deletion type of Nogo-A promoter 2.1kb has no significant effect on Nogo-A promoter activity. Furthermore, we also found that AMPA, which will induced neurotoxicity, did not enhance Nogo-A gene expression. On the contrary, AMPA will down-regulate Nogo-A expression (especially in oligodendrocytes). These findings suggest that the effects of MP and AMPA (200μM in astrocytes and 100μM in oligodendrocytes) combined treatment have higher ability to reduce the expression of Nogo-A in vitro and in vivo.
Similar results could be found in spinal cord injury animal mode. We failed to detect the up-regulation of Nogo-A on the injury site but on adjacent site. Nogo-A expression could be inhibited after MP treatment. Furthermore, MP and AMPA combined treatment could enhance neurite outgrowth in zebrafish system. These results showed the synergistic effect of MP and AMPA, in promoting neurite outgrowth.
Taken together, these results suggest that Nogo-A expression was down-regulated by MP treatment. There may exist certain transcription factors in the Nogo-A promoter of deleted 400bp that modulate MP function on Nogo-A. Moreover, MP and AMPA have synergistic effect on inhibiting Nogo-A expression and promoting neurite growth. The underlining mechanism needs to be further investigated in the future.

中文摘要----------------------------------------------------- I

英文摘要----------------------------------------------------III

前言----------------------------------------------------------1

實驗材料與方法----------------------------------------------- 7

實驗結果-----------------------------------------------------18

討論-----------------------------------------------------25

結論與未來展望---------------------------------------------- 30

參考文獻-----------------------------------------------------31

圖一、Construction of pGL3-Nogo-A promoter expression vector--36
圖二、Analysis of rNogo-A promoter activity in astrocytes --------37
圖三、Expression of Nogo-A gene in astrocytes ------------------38
圖四、Effects of drug treatment on rNogo-A promoter after treatment
----------------------------------------------------39
圖五、Analysis the deletion clones of rNogo-A promoter in astrocytes
----------------------------------------------------41
圖六、Expression of Nogo-A protein --------------------------43
圖七、Expression of Nogo-A gene in oligodendrocytes ------------44
圖八、Effects of drug treatment on rNogo-A promoter activity in OLG
----------------------------------------------------45
圖九、Detection of Nogo-A gene expression in SCI rat -----------46
圖十、Characterization of Nogo-A gene expression at different SCI
sites after MP treatment ------------------------------47
圖十一、Analysis of neurite outgrowth in zebrafish after drug
treatment ----------------------------------------48
圖十二、Analysis of the drug effects of Nogo-A promoter activity at
serum - free condition-------------------------------50
圖十三、Transcription factor motif profiles on Nogo-A promoter
region --------------------------------------------51
附圖一、Comparison of human, mouse and rat Nogo P1 promoter region--------------------------------------------52
附圖二、Immunostaining of GFAP in astrocytes ----------------53

Balazs R, Hack N (1990) Trophic effects of excitatory amino acids in the developing nervous system. Adv Exp Med Biol 268:221-228.
Bracken M, Holford T (1993) Effects of timing of methylprednisolone or naloxone administration on recovery of segmental and long-tract neurological function in NASCIS 2. J Neurosurg 79:500-507.
Bracken M, Shepard M, Collins W, Holford T, Young W, Baskin D, Eisenberg H, Flamm E, Leo-Summers L, Maroon J (1990) A randomized, controlled trial of methylprednisolone or naloxone in the treatment of acute spinal-cord injury. Results of the Second National Acute Spinal Cord Injury Study. N Engl J Med 322:1405-1411.
Brown KM, Wrathall JR, Yasuda RP, Wolfe BB (2004) Glutamate receptor subunit expression after spinal cord injury in young rats. Developmental Brain Research 152:61-68.
Chen MS, Huber AB, van der Haar ME, Frank M, Schnell L, Spillmann AA, Christ F, Schwab ME (2000) Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature 403:434-439.
Christian M, Elizabeth G (2006) Stress and adult neurogenesis. Hippocampus 16:233-238.
Cohen-Cory S, Dreyfus C, Black I (1991) NGF and excitatory neurotransmitters regulate survival and morphogenesis of cultured cerebellar Purkinje cells. J Neurosci 11:462-471.
Collingridge G, Lester R (1989) Excitatory amino acid receptors in the vertebrate central nervous system. Pharmacol Rev 41:143-210.
Diem R, Hobom M, Maier K, Weissert R, Storch MK, Meyer R, Bahr M (2003) Methylprednisolone Increases Neuronal Apoptosis during Autoimmune CNS Inflammation by Inhibition of an Endogenous Neuroprotective Pathway. Journal of Neuroscience 23:6993-7000.
Gasic G, Hollmann M (1992) Molecular neurobiology of glutamate receptors. Annu Rev Physiol 54:507-536.
GrandPre T, Nakamura F, Vartanian T, Strittmatter SM (2000) Identification of the Nogo inhibitor of axon regeneration as a Reticulon protein. Nature 403:439-444.
Hu F, Liu BP, Budel S, Liao J, Chin J, Fournier A, Strittmatter SM (2005) Nogo-A Interacts with the Nogo-66 Receptor through Multiple Sites to Create an Isoform-Selective Subnanomolar Agonist. Journal of Neuroscience 25:5298-5304.
Huber AB, Weinmann O, Brosamle C, Oertle T, Schwab ME (2002) Patterns of Nogo mRNA and Protein Expression in the Developing and Adult Rat and After CNS Lesions. Journal of Neuroscience 22:3553-3567.
Hunt D, Coffin RS, Prinjha RK, Campbell G, Anderson PN (2003) Nogo-A expression in the intact and injured nervous system. Molecular and Cellular Neuroscience 24:1083-1102.
Josephson A, Widenfalk J, Widmer HW, Olson L, Spenger C (2001) NOGO mRNA Expression in Adult and Fetal Human and Rat Nervous Tissue and in Weight Drop Injury. Experimental Neurology 169:319-328.
Liebscher T, Schnell L, Schnell D, Scholl J, Schneider R, Gullo M, Fouad K, Mir A, Rausch M, Kindler D, Hamers F, Schwab M (2005) Nogo-A antibody improves regeneration and locomotion of spinal cord-injured rats. Annals of Neurology 58:706-719.
McDonald J, Johnston M (1993) Excitatory amino acid neurotoxicity in the developing brain. NIDA research monograph 133:185-205.
Mulholland PJ, Self RL, Hensley AK, Little HJ, Littleton JM, Prendergast MA (2006) A 24 h corticosterone exposure exacerbates excitotoxic insult in rat hippocampal slice cultures independently of glucocorticoid receptor activation or protein synthesis. Brain Research 1082:165-172.
Nakanishi S (1992) Molecular diversity of glutamate receptors and implications for brain function. Science 258:597-603.
Nash HH, Borke RC, Anders JJ (2002) Ensheathing Cells and Methylprednisolone Promote Axonal Regeneration and Functional Recovery in the Lesioned Adult Rat Spinal Cord. Journal of Neuroscience 22:7111-7120.
Oertle T, Huber C, van der Putten H, Schwab ME (2003) Genomic Structure and Functional Characterisation of the Promoters of Human and Mouse nogo/rtn4. Journal of Molecular Biology 325:299-323.
Oestreicher AB, De Graan PNE, Gispen WH, Verhaagen J, Schrama LH (1997) B-50, the growth associated protein-43: modulation of cell morphology and communication in the nervous system. Progress in Neurobiology 53:627-686.
Oudega M, Vargas C, Weber A, Kleitman N, Bunge M (1999) Long-term effects of methylprednisolone following transection of adult rat spinal cord. European Journal of Neuroscience 11:2453-2464.
Prinjha R, Moore SE, Vinson M, Blake S, Morrow R, Christie G, Michalovich D, Simmons DL, Walsh FS (2000) Neurobiology: Inhibitor of neurite outgrowth in humans. Nature 403:383-384.
Qian T, Guo X, Levi AD, Vanni S, Shebert RT, Sipski ML (2004) High-dose methylprednisolone may cause myopathy in acute spinal cord injury patients. Spinal Cord 43:199-203.
Reul J, de Kloet E (1985) Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation. Endocrinology 117:2505-2511.
Reul J, Pearce P, Funder J, Krozowski Z (1989) Type I and type II corticosteroid receptor gene expression in the rat: effect of adrenalectomy and dexamethasone administration. Mol Endocrinol 3:1674-1680.
Schoneveld O, Gaemers I, Lamers W (2004) Mechanisms of glucocorticoid signalling. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression 1680:114-128.
Schwab ME (2004) Nogo and axon regeneration. Current Opinion in Neurobiology 14:118-124.
Teng YD, Lavik EB, Qu X, Park KI, Ourednik J, Zurakowski D, Langer R, Snyder EY (2002) Functional recovery following traumatic spinal cord injury mediated by a unique polymer scaffold seeded with neural stem cells. Proceedings of the National Academy of Sciences 99:3024-3029.
Tsai S-Y, Yang L-Y, Wu C-H, Chang S-F, Hsu CY, Wei C-P, Leu S-J, Liaw J, Lee Y-H, Tsai M-D (2006) Injury-induced Janus kinase/protein kinase C-dependent phosphorylation of growth-associated protein 43 and signal transducer and activator of transcription 3 for neurite growth in dorsal root ganglion. Journal of Neuroscience Research 85:321 - 331.
Tsai SY, Chiu PY, Yang CP, Lee YH (2002) Synergistic effects of corticosterone and kainic acid on neurite outgrowth in axotomized dorsal root ganglion. Neuroscience 114:55-67.
Wang KC, Kim JA, Sivasankaran R, Segal R, He Z (2002a) p75 interacts with the Nogo receptor as a co-receptor for Nogo, MAG and OMgp. Nature 420:74-78.
Wang X, Chun S-J, Treloar H, Vartanian T, Greer CA, Strittmatter SM (2002b) Localization of Nogo-A and Nogo-66 Receptor Proteins at Sites of Axon-Myelin and Synaptic Contact. Journal of Neuroscience 22:5505-5515.
Wang Y, Tseng G (2004) Spinal axonal injury transiently elevates the level of metabotropic glutamate receptor 5, but not 1, in cord-projection central neurons. Journal of Neurotrauma 21(4):479-489.
Westerfield, M. (1995) The Zebrafish Book. 3rd end. Eugene, OR: University of Oregon Press.
Zipfel G, Babcock D, Lee J, Choi D (2000) Neuronal apoptosis after CNS injury: the roles of glutamate and calcium. J Neurotrauma 17:857-869.