臺灣博碩士論文加值系統

我們研究長期給予1-甲基-4-酚基-1,2,3,6,-四氫嘌呤 (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP)此種會選擇性導致dopa-minergic neuron退化的藥物，想知道此藥物是否會對下視CRF及ACTH調控路徑產生影響？因此，我們連續7天給予C57BL/6J mice MPTP，在停止給藥後之第一天、第三天、第七天、第十四天以及第三十天犧牲老鼠取其週邊血，分別利用酵素免疫分析(Enzyme-linked-immuno sorbent assay, ELISA) 及放射免疫分析法 (Radio-immunoassay, RIA) 的方法分別偵測小黑鼠血中的促腎上皮質釋放激素及ACTH的濃度。我們發現長期給予MPTP並不會影響血中CRF的澧度，但在停藥後第一天會短暫性增加ACTH在血中的濃度。因此，由此結果可知，在MPTP的打藥過程中，有可能也會影響到CRF的表現，因而導致停藥後ACTH仍然會有短暫性上升。接下來，我們為了要偵測MPTP給藥後對神經細胞造成的損傷，是否會影響到NMDA受體的表現?因此，我們利用西方點墨法，在連續7天給予動物MPTP後，分別在停藥後第1、3、7、14、30天，去偵測大腦皮質、海馬迴、紋狀體、下視丘上的NMDA各種受體多亞型蛋白。結果我們發現，MPTP給藥後，所引起各種NMDA受體的變化十分複雜，但與過去本實驗室以[3H]TCP ligand binding，所偵到會有短暫性抑制NMDA受體的數目結果並不一致。另外，我們亦偵測酪胺酸羥化酵素(Tyrosine hydroxylase,TH )、細胞骨架蛋白(spectrin)在腦中的含量，利用此兩種蛋白質的表現量，來看長期給予MPTP對多巴胺神經元所造成毒性的存在。我們發現，在停藥後的30天內，TH在紋狀體有慢慢回復的情形，配合spectrin在細胞膜上的表現，我們也發現spectrin也有逐漸回復的情形。不過，我們在海馬迴中並未偵測到TH的表現量，而其spectrin在細胞膜上的表現量亦無降低的情形。終其這些結果，我們發現，長期給予MPTP對紋狀體系統會有很明顯的生化損傷，表現在TH及spectrin上。但MPTP給藥後，對NMDA受體的表現，其結果尚無法得到一個結論。

We have determined the effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on the releasing corticotropin releasing factor (CRF) and adrenocoricotrophic hormone (ACTH) by quantifying the plasma level CRF and ACTH using radioimmuno-assay (RIA) and enzyme-linked-immuno sorbent assay (ELISA) in the C57BL/6J mice with 7 days treatment of MPTP. We found that MPTP treatment did not affect the plasma level of CRF, but transiently increase in the plasma level of ACTH at 1st day after continuation MPTP treatment. This result suggested that chronic MPTP treatment might affect CRF-ACTH pathway and degenerate the dopaminergic system. However, this effect might occur before continuation of MPTP treatment. We also determine the NMDA subunits expression with cortex, hippocampus, striatum and hypothalamus after continuation MPTP treatment. We found very complicated fluctuation of NMDA receptor subunits expression in different brain region. However, there was no change of NMDA receptor subunits in the cortex. In the hippocampus, most of subunits were unevenly increased. There was complicated fluctuation on the NMDA receptor subunits expression in the striatum and hypothalamus. We also determined the neurotoxity of MPTP on the neuronal cells by examining the expression of tyrosine hydroxylase (TH) and neurodentritic spectrin on membrane and its degradation product. We found that in the striatum, there was gradually reverse of the TH after continuous MPTP treatment. The level of the TH on the 30th days after MPTP treatment was not significantly different to that of control group in the cortex. In the striatum, accompanied with this phenomena, the expression of spectrin on the membrane, there was a transit decrease at the 1st, 3th day after MPTP treatment but than become no signification to control group 7 day after MPTP treatment. This result indicated that MPTP treatment could induce striatum neuron degernation.

目次
中文摘要…………………………………………………………………i
英文摘要…………………………………………………………………iii
表目錄……………………………………………………………………v
圖目錄……………………………………………………………………vi
第一部分 長期給予MPTP對小黑鼠血中促腎上腺皮質素
(ACTH)以及促腎上腺皮質釋放激素(CRF)表現的影響
壹、前言…………………………………………………………………1
一、 巴金森氏症的介紹………………………………………………1
二、 巴金森氏症的致病原因…………………………………………2
(一)腦中的多巴胺神經路徑…………………………………………3
(二) MPTP的動物模式…………………………………………………4
三、 促腎上皮質釋放激素、促腎上腺皮質素與巴金森氏症………5
貳、實驗材料與方法……………………………………………………9
一、 實驗材料…………………………………………………………9
(一) 動物品種來源……………………………………………………9
(二) 實驗藥品…………………………………………………………9
(三) 儲備溶液…………………………………………………………9
(四) 實驗儀器…………………………………………………………10
二、實驗方法…………………………………………………………10
(一)動物給藥模式……………………………………………………10
(二)組織處理…………………………………………………………10
(三)Enzyme-Linked-Immuno Sorbent Assay (ELISA)……………11
(四)放射免疫分析法…………..……………………………………12
(五) 統計方法………………………………………………………12
參、結果………………………………………………………………14
肆、討論………………………………………………………………15
伍、結論………………………………………………………………18
第二部分 長期給予MPTP對小黑鼠腦中NMDA受體多亞型
蛋白以及細胞骨架蛋白(spectrin) 表現的影響
壹、前言………………………………………………………………19
一、 NMDA受體與巴金森氏症的關係…………………………………19
(一)Glutamate受體與巴金森氏症的關係……………………………19
(二)NMDA受體與巴金森氏症的關係…………………………………20
(三)NMDA受體的介紹…………………………………………………21
二、Spectrin與Cell Degeneration的關係………………………23
貳、實驗材料與方法…………………………………………………27
一、實驗材料…………………………………………………………27
(一)動物品種來源……………………………………………………27
(二)實驗藥品…………………………………………………………27
(三)儲備溶液…………………………………………………………31
(四)實驗儀器…………………………………………………………31
二、實驗方法…………………………………………………………33
(一)動物給藥模式……………………………………………………33
(二)組織處理…………………………………………………………33
(三)西方墨點法………………………………………………………33
(四)數據處理及分析…………………………………………………37
(五)統計方法…………………………………………………………38
參、結果………………………………………………………………39
肆、討論………………………………………………………………45
伍、結論………………………………………………………………49
參考文獻………………………………………………………………50

參考文獻
Allen B. King (1969) The effect of exogenous dopamine on ACTH secretion. Proceedings of the society for experimental biology and medicine. 130: 445-447.
Bennett V., Davis J. and Fowler W. E. (1982) Brain spectrin , a membrane-associated protein related in structure and formation to erythrocyte spectrin. Nature 299:126-131.
Bonuccelli U., Del Dotto P., Piccini P., Beghe F., Corsini G.U., Muratorio A. (1992) Dextromethorphan and Parkinsonism. Lancet 340:53.
Carlsson M., Carlsson A.,(1990)Interactions between glutamatergic and mono-aminergic systems within the basal ganglia-implications for schizophrenia and Parkinson's disease. Trends Neurosci. 13:272-277.
Chiueh C. C., Markey S. P., Burns R. S., Johannessen J. N., Jacobowitz D. M. and Kopin J. (1984) Neurochemical and behavioral effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in rat, guinea pig and monkey. Psychopharmac. Bull. 20 : 548-553.
Choi S.W.(1988) Glutamate neurotoxicity and disease of the nervous system. Neuron 1: 623-634.
Coyle J.T., Puttfarcken P. (1993) Oxidative stress, glutamate, and neurodegenerative disorders. Science 262: 689-695.
Davis G.C., Williams A.C., Marley S.P., Ebert M.H., Calne E.D., Reichert C.M. and Kopin I.J. (1979) Chronic Parkinsonism secondary to intravenous injection of meperidine analogues. Psychiatry Res.1: 249-254.
De Souza EB., Bissette G., Whitehouse, PJ, Powers RE., Price DL., Vale WW., Nemeroff CB. (1990) Abnormalities in corticotropin-releasing factor (CRF) in neurodegenerative diseases . In ‘Corticotropin-Releasing Factor: Basic and Clinical Studies of a Neuropeptide.’ De Souza EB., Nemeroff CB (eds): New York: CRC Press,335-350.
Defrance J.F., Sike R.W. and Chronister R.B. (1985) Dopamine action in the nucleus accumbens. J. Neurophysiologhy 54:1568-1577.
Evans R. H., Francis A. A., and Watkins J. C.(1977) Selective antagonism by Mg2+ of amino acid-induced depolarization of spinal neurons. Experientia (Basel) 33: 489-491.
Fornai F., Vaglini F., Maggio R., Bonuccelli U., Corsini G.U. (1997) Species differences in the role of excitatory amino acids in experimental parkinsonism. Neurosci. and Biobehavioral Rev. 21(4): 401-415.
Fukunaga K, Soderling TR, Miyamoto E(1992) Activation of Ca2+/calmodulin-dependent protein kinase Ⅱ and protein kinase C by glutamate in cultured rat hippocampal neurol neurons. J.Biol Chem 267: 22527-22533.
Ganong W.F.: Review of medical physiology. 15th ed. Appleton and Lange Press, New York, 1993.
Glenney J. R., Glenney P., Jr and Weber K. (1982) Erythroid spectrin ,brain fodrin, and intestinal brush border proteins (Tw-260/240) are related molecules containing a common calmodulin-binding subunit bound to a variant cell type-specific subunit. Pro. Nat. Acadeny of Sci. U.S.A. 79: 4002-4005.
Gibb W.R. and Lees A.J. (1988) The relevance of Lewy body to the pathogesis of idiopathic Parkinson’s disease. J Neurosurg Psychiatry 51:745-752.
Giulian D (1992) Brain inflammatory cells, neurotoxins, and acquired immuno-deficiency syndrome. In Excitatory amino acids,(ed. R. P. Simon)229-234. Thieme Medical Publislers, Inc., New York.
Coyle J. T., and Puttfarcken P. (1993) Oxidative stress, glutamate, and neuro-degenerative disorders. Science 262: 689-695.
Doble A. (1995) Excitatory amino acid receptors and neurodegeneration. Therapie 50 : 319-337.
Greenamyre J.T. and O'Brien C. F. (1991) N-methyl-D-asparate antagonists in the treatment of Parkinson's disease. Archives of Neurology 48:977-981.
Heikkila R. E., Hess A. and Duvoisin R. C. (1984) Dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice. Science 224:1451-1453.
Hollmann M., Heinemann S. (1994) Cloned glutamate receptors. Annu. Rev. Neurosci. 17:31-108.
Horellou P. and Mallet J. (1997) Gene therapy for Parkinson’s disease. Molecular Neurobiology 15:241-256.
Huang C.C. and Lee E.H.Y. (1995) Alteration of corticotropin-releasing factor immunoreactivity in MPTP-treated rats. J Neurosci. 41:471-480.
Ishii T., Moriyoshi K., Sugihara H., Sakurada K., Kadotani H., Yokoi M., Akazara C., Shigemoto R., Mizuno N., Masu M. and Nakanishi S. (1993) Mokecukar characterization of the family of the N-methyl-D-aspartate receptor subunits. J. Biol. Chem. 268: 3728-3733.
Irwin I., Langston J.W. (1985) Selective accumulation of MPP+ in the substantia nigra : a key to neurotoxicity ? Life Sci. 36:207.
Jackson-Lewis, Jakowec M. Burke R.E. (1995) Time course and morphology of dopaminergic neuroanl death caused by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Neurodegeneration 4；257-269.
Jenner P. (1998) Oxidative mechanisms in nigral cell death in Parkinson’s disease. Movement Disorders 13 suppl 1: S24-S34.
John W. M.D. and Michael V. J. (1990) Physiological and pathophysiological roles of excitatory amino acids during central nervous system development. Brain Res. Rev. 15:41-70.
Jolkkonen J., Hartikainen P., Soikkeli R., Bissette G., Nemeroff C., Riekkinen P. (1991) A correlation study of CRF neuropeptides in Alzheimer’s and Parkinson’s disease. Neuropeptides. 19:97-102.
Klockgether T. and Turski L. (1990) NMDA antagonists potentiate antiparkinsonian actions of L-dopa in monoamine-depleted rats. Annu. of Neurology 28: 539-546.
Kloog Y., Haring R. and Sokolovsky M. (1988)Kinetic characterization of the phencycline N-methyl-D-asparate receptor interaction: evidence for a steric blockade of the channel. Biochemistry 27: 843-848.
Kupsch A., Löschmann P. A., Sauer H., Arnold G., Renner P., Pufal M., Wachtel H., Bruggencate G., Oertel W. H. (1992) Do NMDA receptor receptor antagonists protect against MPTP-toxicity? Biochemical and immunocytochemical analyses in black mice. Brain Res. 592:74-83.
Kutsuwada T., Kashiwabuchi N., Moro H., Sakimura K., Kushiya E., Araki K., Meguro H., Masaki H., Kumanishi T., Arakawa M and Mishina M. (1992). Molecular diversity of the NMDA receptor channel. Nature, 358, 36-41.
Langston J.W., Ballard P., Tetrud J.W. and Irwin I. (1983) Chronic parkinsonism in human due to a product of meperidine analogues synthesis. Science 219:979- 980.
Langston J.W. (1985) Mechanism of MPTP toxicity: more answers, more questions ,Trends Pharmacol. Science 6 : 375.
Langston J.W. (1996) The etiology of Parkinson’s disease with emphasis on the MPTP story. Neurology 473:S153-S160.
Larson A. A. and BetizA.J.(1988) Glycine potentiates strychanine-induced convulsion: role of NMDA receptors. J. Neurosci. 8 : 3822-3826.
Lazarides E and Nelson W.J.(1983) Erythrocyte and brain-cytoskeletal domains in neurons. Science 220: 1295-1296.
Lee Eminy H.Y. (1988) Neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetra- hydropyridine on striatal biopterin in BALB/c mice. Neurosci. Letters 88:297-302.
Macdermott A. B., Mayer M. L., Westbrool G. L., Smith S. J., and Barker J. L. (1986) NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurons. Nature (Lond.) 321: 519-522.
Manfred G., Peter R., Moussa B.H.Youdim (1991) MPTP mechanisms of neuro- toxicity and their implications for Parkinson’s disease. Eur. J. Pharmacol. 208:273-286.
Mayer M. L. and Westbrool G. L. (1987a) The physiology of exicitatory amino acids in the vertebrate central nervous system. Progress in Neurobiology 28:197-276.
Mayer M. L., MacDermott, A.B., Westbrool G. L., G.L., Smith, S. J., and Barker, J. L. (1987b). Agonist- and voltage-gated calcium entry in cultured mouse spinal cord neurones under voltage clamp measured using arsenazo Ⅲ. J. Neurosci. 7 : 3230-3244.
Mayer M. L., Viklicky L.Jr., and Sernegor E. (1989) A physiologist's view of the N- methyl-D-asparate receptor: an allosteric ion channel with multiple regulatory sites. Drug DeV research 17: 263-280.
McDermott A. B., Dale N. (1987)Receptors, ion channels and synaptic potentials underlying the integrative actions of exitatory amino acids. Trends Neurosci.10: 280-284.
Merchenthaler I., S Vigh, P. Petrusz and A.V. Schally (1982) Immunocytochemical localization of corticotropin-releasing factor (CRF) in rat brain. Am. J. Anat. 165:385.
Mizobuchi M., Hineno T., Kakimoto Y.,and Hirratani K. (1993) Increase of plasma adrenocorticotrophin and cortisol in 1-methyl-4-phenyl-1,2,3,6-tetrahdro- pyridine (MPTP)-treated dog. Brain Res.. 612: 319-321.
Mizuno Y., Sone N., Saitoh T. (1987) Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine and 1-methyl-4-Phenyl- pyridinium ion on activities of the enzymes in the electron transport system in mouse brain. J. Neurochem. 48: 1787.
Montastruc J.L., Rascol O., Senard J.M., Rascol A. (1992) A pilot study of N-methyl- D- aspartate(NMDA) antagonist in Parkinson’s disease. J. Neurol. Neurosurg. Psychiatr. 55: 630-631.
Monyer H., Sprengel R., Schoepfer R., Herb A., Higuchi M., Lomeli H., Burnashev N., Sakmann B., Seeburg P.H. (1992) Heteromeric NMDA receptor: molecular and functional distinction of subtypes. Sci. 256:1217-1221.
Mori H. and Mishina M. (1995) Structure and function of the NMDA receptor channel. Neuropharmacol. 34: 1219-1237.
Nakanishi S. (1992) Molecular diversity of glutamate receptors and implications for brain function. Science 258:597-603.
Nakanishi N., Axel R., and Shneider N. A. (1992a) Alternative splicing generates functionally distinct N-methyl-D-aspartate receptors. Proceedings of National Academy of Sci., USA 89；8552-8556.
Nakanishi S., Masu M. (1994) Molecular diversity and functions of glutamate receptors. Annu. Rev. Biophys. Biomol. Struct. 23: 319-348.
Nakanishi S., Nakajima Y., Masu M., Ueda Y., Nakahara K., Watanabe D., Yamaguchi S., Kawabata S. (1998) Glutamate receptors : brain function and signal transduction. Brain Res. Rev. 26:230-235.
Nicklas W. J., Vyas I., Heikkila R. E. (1985) Inhibition of NADH-linked oxidation in brain mitochondria by 1-methyl-4-Phenyl-pyridine, a metabolite of the neuro- toxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine. Life Sci. 36:2503.
Nowak L., Bregestovski P., Ascher P., Herbet A., and Prochiantz A.(307) Nature 307: 462-465.
Olianas MC., Onali P. (1989) Stimulation of synaptosomal dopamine synthesis by corticotropin-releasing factor in rat striatum : role of Ca2+ dependent mechanisms. Eur. J. Pharmacol 166(2): 165-174.
Ossowska K. (1994) The role of excitatory amino acids in experimental models of Parkinson’s disease. J. Neural. Transm. (P-D Sect) 8:39-71.
Perry T. L.and Hansen S. (1981) Amino acid abnormalities in epileptogenic foci. Neurology (New York) 31:872-876.
Perry T. L., Krieger C., Hansen S and Eisen A(1990) Amyotrophic lateral sclerosis : amino acid levels in plasma and cerebrospinal fluid. Annals of Neurology 28: 12-17.
Ramsay R.R., McKeown K.A., Johnson E.A., Booth R.G., Singer T.P. (1987) Inhibition of NADH oxidation by pyridine derivatives. Biodhem. Biophys. Res. Commun. 146:53.
Pileblad E., Carlsson A. (1985)Catecholamine-uptake inhibitors prevent the neuro- toxicity of 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine(MPTP) in mouse brain. Neuro-pharmacol. 24 : 689-692.
Ricaurte G. A., Langston J.W., DeLanney L. E., Irwin I., Brooks J. D. (1985) Dopamine uptake blockers protect against the dopamine depleting effect of 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine(MPTP) in the mouse striatum. Neurosci. Lett. 59: 259-264.
Riederer B.M., Zagon I.S., Goodman S.R. (1987) Brain spectrin (240/235) and Brain spectrin(240/235E) : differential expression during mouse brain development. J. Neurosci. 7(3): 864-874.
Rivier C., Brownstein M., Spiess J., Rivier J., Vale W. (1981) In vivo corticotropin releasing factor-induced secretion of adrenocorticotropin, β-endorphin and corticosterone. Endocrinology 110: 272-278.
Robert S. and Noszek J.C. (1988) Excitatory amino acids activate calpainⅠ and induce structural protein breakdown in vivo. Neuron 1: 279-287.
Serge P., Veernice J.L. (1998)Mechanisms of MPTP toxicity. Movement Disorders 13 suppl 1 : S35-S38.
Spencer P.S., P.B. Nunn, H. Hungon, J. C. Ludolph, S. M. Ross, D. N. Roy and R. C. Robertson (1987) Guam amyotrophic lateral sclerosis-parkinsonism-dementia liked to a plant neurotoxin. Science 237 : 517-522.
Sundstrom E., Jonsson G. (1985) Pharmacological interference with the neurotoxic action of 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine(MPTP) on central catecholamine neurons in the mouse. Eur. Pharmacol. 110: 293-299.
Steven R. and Ian S. (1984) Brain spectrin: A review. Brain Res. Bull. 13:813-832.
Tingley W. G., Roche K. W., Thompson A. K., and Huganir R. L. (1993) Regulation of NMDA receptor phosphorylation by alternative splicing og the C-terminal domain. Nature 364:70-73.
Turski L., Bressler K. Rettig, K-A., amd wachtel, H. (1991) Protection of substantia nigra from MPP neurotoxicity by N-methyl-D-asparate antagonists. Nature 349: 414-418.
Ułas J., Weihmuller FB., Brunner LC., Joyce JN., Marshall JF., and Cotman CW. (1994) Selective increase of NMDA-sensitive glutamate binding in the striatum of Parkinson's disease, Alzheimer's disease, and mixed autoradio-graphic study .J. Neurosci. 14 (11): 6317-6324.
Vale W. Spiess J. Rivier J. (1981) Characterization of a 41-residue ovine hypo- thalamic peptide that stimulates secretion of corticotropin and β-endo- rphin. Science 213: 1394-1397.
Vyas I., Heikkila R.E., Nicklas W. J. (1986) Studies on the neurotoxicity of 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine: inhibition of NAD-linked substrate oxidation by its metabolite, 1-methyl-4-Phenyl-pyridinium . J. Neurochem. 46 : 1501.
Weihmuller F.B. Ułas J, Nguyen L., Cotman CW., Marshall JF (1992) Elevated NMDA receptors in Parkinsonism striatum. Neuroport 3 :977-980.
Wüllner U., Brouillet E., Young A.B. and Penney J.B. (1993) Glutamate receptor binding sites in MPTP-treated mice. Exp. Neurology. 121: 284-287.
Zagon I.S., Higbee R., Riederer B.M., Goodman S.R. (1986) Spectrin subtypes in mammalian brain: an immunoelectron microscopic study. J. Neurosci. 6: 2977-2986.