甲基安非他命 (Methamphetamine) 為一個常見的濫用藥物，被發現對神經系統具有毒性，其神經傷害的機轉較為複雜，目前正陸續被釐清中。目前確定，甲基安非他命傷害的機轉包括造成粒線體及內質網的氧化壓力增加，並藉由csapase依存或非caspase依存路徑的一連串活化造成細胞凋亡。本論文利用培養大白鼠皮質神經細胞並給予甲基安非他命的傷害模式，來篩選我們認為對甲基安非他命可能具有神經保護功能，且臨床上容易使用的六個藥物(維他命E、VPA、鋰鹽、LC、FK506及bilobalide)進行篩選，並探討及比較其保護機轉間的差異與功效。實驗結果顯示，給予甲基安非他命6小時可造成細胞內的自由基釋放，給予24小時會粒腺體膜電位下降，給予48小時則會造成初級大腦皮質細胞之細胞凋亡。在神經保護製劑篩選方面，我們發現前處理維他命E及VPA可保護甲基安非他命導致大白鼠皮質神經細胞的細胞凋亡，以及抑制細胞內的自由基增加；此外，VPA及高濃度之維他命E能回復細胞粒線體膜電位。而前處理鋰鹽並不能保護甲基安非他命導致大白鼠皮質神經細胞的細胞凋亡；前處理FK506能抑制甲基安非他命造成的大白鼠皮質神經細胞的細胞凋亡，及粒線體膜電位的下降，但它並不能降低甲基安非他命所造成的細胞自由基增加；LC與bilobalide雖能抑制甲基安非他命造成的大白鼠皮質神經細胞的細胞凋亡及細胞自由基的增加，但對於粒線體膜電位的下降並不能顯著回復。綜合以上，在大腦皮質神經細胞給予甲基安非他命造成細胞損傷的模式中，維他命E、 VPA、FK506與LC雖然對細胞的保護機轉略為不同，但都顯示能保護甲基安非他命造成的細胞凋亡，而bilobalide及鋰鹽對大腦皮質神經細胞受甲基安非他命造成的神經細胞損傷無保護作用。在大腦皮質細胞受到甲基安非他命造成的細胞凋亡傷害機轉中，粒線體功能的維持與抑制細胞凋亡似乎有重要關聯，而與清除甲基安非他命造成之自由基釋放似乎較關聯較間接。

Methamphetamine (METH) is a popular drug of abuse with the neurotoxic mechanisms that are complicated and remains to be studied. We have used METH to cause cortical neuron apoptosis model, and then investigated the neuroprotective effects of six potential neuroprotective agents including vitamine E (vit E), valproic acid (VPA), LiCl, L-Carnitine(LC), FK506 and bilobolide.
According to our findings, the METH caused the intercellular ROS release (6 hours), decreased the mitochondrial membrane potential (24 hours), and caused cortical neuron culture apoptosis (48 hours). We further showed that vit E and VPA can prevent cell apoptosis and ROS release induced by METH. Besides, vit E and high dose of VPA can prevent the decrease of mitochondrial membrane potential induced by METH. The cell apoptosis induced by METH can not be prevented by LiCl. FK506, which is a calcineurin inhibitor, prevented the METH-induced cell apoptosis and mitochondrial membrane potential decreasing. However, FK506 can not prevent ROS increasing induced by METH. LC and bilobolide can prevent call apoptosis and ROS increasing induced by METH, but can not recover the mitochondrial membrane potential changed by METH.
In summary, the neuroprotective mechanisms of those drugs are different. Our studies showed that vit E, VPA, FK506, LC and bilobolide can protect METH-induced neurotoxicity.

目錄
                 頁次
目錄………………………………………………………………………I
圖次………………………………………………………………………II
中文摘要…………………………………………………………………III
英文摘要…………………………………………………………………V
第一章 緒言…………………………………………………………..1
第二章 研究目的…………………………………………………….27
第三章 方法與材料………………………………………………….28
第四章 結果………………………………………………………….36
第五章 討論………………………………………………………….55
第六章 結論………………………………………………………….66
第七章 參考文獻…………………………………………………….98

第七章、參考文獻

Abid-Essefi S, Baudrimont I, Hassen W, Ouanes Z, Mobio TA, Anane R, Creppy EE, Bacha H. DNA fragmentation, apoptosis and cell cycle arrest induced by zearalenone in cultured DOK, Vero and Caco-2 cells: prevention by Vitamin E. Toxicology. 5;192(2-3):237-48, 2003.
Ahlemeyer B. and Krieglstein J. Neuroprotective effects of the Ginkgo biloba extract. In: Phytomedicine of Europe: Chemistry and Biological activity. ACS Symposium Series. 691:210–220, 1998.
Virmani A, Gaetani F, Imam S, Binienda Z, and Ali S. Possible Mechanism for the Neuroprotective Effects of L-Carnitine on Methamphetamine-Evoked Neurotoxicity. Ann. N.Y. Acad. Sci.993:197–207, 2003.
Ali SF, Newport GD, and Slikker W. Methamphetamine-induced dopaminergic toxicity in mice. Role of environmental temperature and pharmacological agents. Annals of the New York Academy of Sciences. 801:187-98, 1996.
Bennett BA, Hollingsworth CK, Martin RS, and Harp JJ. Methamphetamine-induced alterations in dopamine transporter function. Brain Research. 782(1-2):219-27, 1998.
Borner C and Monney L. Cell Death Differ. 6:497–507, 1999.
Bowyer JF, Holson RR, Miller DB, and O'Callaghan JP. Phenobarbital and dizocilpine can block methamphetamine-induced neurotoxicity in mice by mechanisms that are independent of thermoregulation. Brain Research. 919(1):179-83, 2001.
Braestrup C. Biochemical differentiation of amphetamine vs methylphenidate and nomifensine in rats. Journal of Pharmacy &Pharmacology. 29(8):463-70, 1977.
Bolanos JP. Almeida A. Stewart V. Peuchen S. Land JM. Clark JB. Heales SJ. Nitric oxide-mediated mitochondrial damage in the brain: mechanisms and implications for neurodegenerative diseases. Journal of Neurochemistry. 68(6):2227-40, 1997.
Bunney BS, Walters JR, Kuhar MJ, Roth RH, and Aghajanian GK. D & L amphetamine stereoisomers: comparative potencies in affecting the firing of central dopaminergic and noradrenergic neurons. Psychopharmacology Communications. 1(2):177-90, 1975.
Ahlemeyer B. and Krieglstein J. Review Neuroprotective effects of Ginkgo biloba extract. CMLS, Cell. Mol. Life Sci. 60 :1779-1792, 2003.
Cai J, Yang J, and Jones DP . Mitochondria control of apoptosis：The role of cytochrome c. Biochim Biophys Acta. 1366:139-149, 1998.
Callahan LA, Stofan DA, Azweda LI, Nethery DE, and supinski GS. Free radical alter maximal diaphragmatic mitochondrial oxygen consumption in endotoxin-induced sepsis. Free Radic Bio Med. 30:129-138, 2001.
Chan SL, Mattson MP. Caspase and calpain substrates: roles in synaptic plasticity and cell death. Journal of Neuroscience Research. 58(1):167-90, 1999 .
Chuang D-M, Chen R-W, Chalecka-Franaszek E, Ren M. andPeter Leeds. Review Article Neuroprotective effects of lithium in cultured cells and animal models of diseases. Bipolar Disorders. 4:129-136, 2002.
Cho AK, Wright J. Pathways of metabolism of amphetamine and related compounds. Life Sciences. 22(5):363-72, 1978.
Clendenin WW. Amphetamine Dependence. Clinical Pharmacology and Drug Dependence. 215-238, 1984.
Cohen G. Enzymatic/nonenzymatic sources of oxyradicals and regulation of antioxidant defenses. Annals of the New York Academy of Sciences. 738:8-14, 1994.
Davidson C, Gow AJ, Lee TH, and Ellinwood EH. Methamphetamine neurotoxicity: necrotic and apoptotic mechanisms and relevance to human abuse and treatment. Brain Research - Brain Research Reviews. 36(1):1-22, 2001.
Dawson TM, Steiner JP, Dinerman JL, Uhl GR, Snyder SH, Immunosuppressant, FK506, enhances phosphorylation of nitric oxide synthase and protects against glutamate neurotoxicity. Proc. Natl. Acad. Sci. U. S. A. 90, 9808– 9812, 1993.
DeFeudis FV. Ginkgo biloba extract (EGb 761): pharmacological Activities and Clinical Applications, Elsevier, Paris,1991.
Desagher S and Martinou JC. Mitochondria as the central control point of apoptosis. Trends Cell Biol. 10, 369–377, 2000.
Deng X, Cadet JL. Methamphetamine administration causes overexpression of nNOS in the mouse striatum. Brain Research. 851(1-2):254-7, 1999.
Earnshaw WC, Martins LM, and Kaufman SH. Mammalian caspases: structural, activation, substrates, and functions during apoptosis. Annu. Rev. Biochem. 68:383-424, 1999.
Espelin DE, Done AK. Amphetamine poisoning. Effectiveness of chlorpromazine. New England Journal of Medicine. 278(25):1361-5, 1968.
Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, and Nagata S. A caspase-activated DNase during apoptosis, and its inhibitor ICAD. Nature 391：43-50, 1998.
Ferri KF, Jacotot E, Blanco J, Este JA, and Kroemer G. Mitochondrial control of cell death induced by proteins encoded by HIV-1. Annals New York Acad. Sci. 926:149-164, 2000.
Fukunaga T, Mizoi Y, Adachi J. Methamphetamine-induced changes of peripheral catecholamines: an animal experiment to elucidate the cause of sudden death after methamphetamine abuse. Nippon Hoigaku Zasshi - Japanese Journal of Legal Medicine. 41(4):335-41, 1987.
Fumarola C and Guidotti G. Stress-induced apoptosis：toward symmetry with receptor-mediated cell death. Apoptosis 9 :77-82, 2004.
Green DR and Reed JC. Mitochondria and apoptosis. Science 281: 1309-1312, 1999.
Gunter TE and Pfeiffer DR：Mechanisms by which mitochondria transport calcium. Am. J. Physiol 258:C755-C786, 1990.
Forloni G, Angeretti N, and Smiroldo S. Neuroprotective Activity of Acetyl-L-Carnitine: Studies In Vitro. Journal of Neuroscience Research. 37 : 92-96, 1994.
Heneka MT, Loschmann PA, Gleichmann M, Weller M, Schulz JB, Wullner U, and Klockgether T. Induction of nitric oxide synthase and nitric oxide-mediated apoptosis in neuronal PC12 cells after stimulation with tumor necrosis factor-alpha/lipopolysaccharide. Journal of Neurochemistry. 71(1):88-94, 1998.
Hengartner MO. The biochemistry of apoptosis. Nature. 407 :770-776, 2000.
Takahashi H, Kosaka N and Nakagawa S. α–Tocopherol Protects PC12 cells From Hyperoxia-Induced Apoptosis. Journal of Neuroscience Research. 52 :184-191, 1998.
Huang NK, Wan FJ, Tseng CJ, and Tung CS. Nicotinamide attenuates methamphetamine-induced striatal dopamine depletion in rats. Neuroreport. 8(8):1883-5, 1997.
Karch SB. The pathology of drug abuse. CRC press in the United States. 166-218, 1993.
Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer. 26(4):239-57, 1972.
Kerr JF, Winterford CM, Harmon BV. Apoptosis. Its significance in cancer and cancer therapy. Cancer. 73(8):2013-26, 1994.
King GR and Ellinwood EH. Amphetamine and other stimulates. 1992.
Krieglstein J, Ausmeier F, El-Abhar H, Lippert K, Welsch M, Rupalla K et al. Neuroprotective effects of Ginkgo biloba constituents. Eur. J. Pharm. Sci. 3: 39–48, 1995.
Kroemer G. The proto-oncogene Bcl-2 and its role in regulating apoptosis . Nat Med. 3：614-620, 1997.
Leppa S, Saffrich R, Ansorge W, and Bohmann D. Differential regulation of c-Jun by ERK and JNK during PC12 cell differentiation. EMBO Journal. 17(15):4404-13, 1998.
Zhou L-J and Zhu X-Z. ROS-Induced Apoptosis and Protective Effect of Bilibalide. JPED 293 :982-988, 2000.
Ly JD, Grubb DR, and Lawen A. 2003. The mitochondria membrane potential (ΔΨm) in apoptosis. a updata. Apoptosis 8:115-128, 2003.
Majno G, Joris I. Apoptosis, oncosis, and necrosis. An overview of cell death. American Journal of Pathology. 146(1):3-15, 1995.
Mario PR, White ER, McDonald SA, Hill JM, Jeffcoat AR and Cook CE. Clinical effects of methamphetamine vapor inhalation. Life Science. 49:953-959, 1991.
Matthew G Annis, Naoufal Zamzami, Weijia Zhu, Linda Z Penn, Guido Kroemer,Brian Leber, and David W Andrews. Endoplasmic reticulum localized Bcl-2 prevents apoptosis when redistribution of cytochrome c is a late event. Oncogene. 20: 1939-1952, 2001.
Jeong MR, Hashimoto R, Senatorov VV, Fujimaki K, Ren M, Lee MS, Chuang D-M. Valproic acid, a mood stabilizer and anticovulsant, protects rat cerebral cortical neurons from spontaneous cell death: a role of histone deacetylase inhibition. FEBS Letters. 542:74-78, 2003.
Murphy AN, Fisjum G, and Beal MF. mitochondria in neurodegeneration bioenergetic function in cell life and death. J Cereb Blood Flow Metab. 17:815-832, 1999.
Virmani MA, Rossi S, Conti R, Spadoni A, E and Calvani M. Pharmacological Research. 33:19-27, 1996.
Virmani MA, Biselli R, Spadoni A, Rossi S, Corsico N, Calvani M, and Fattorossi A. Pharmacological Research. 32, 383-389, 1995.
Gurvich N and Klein PS. Lithium and valproic acid: parallels and contrasts in diverse signaling contexts. Pharmacology and Therapeutics. 96:45-66, 2002.
Nishino H, Fujimoto I, Shimano Y, Hida H, Kumazaki M and Fukuda A. 3-Nitropropionic acid produces striatum selective lesions accompanied by iNOS expression. J. Chem. Neuroanat. 10, 209– 212,1996.
Nutt LK, pataer A, Pahler J, Fang B, Roth J, Mcconkey DJ, and Swisher SG. Bax and Bak promote apoptosis by modulating endoplasmic reticular and mitochondrial Ca2+ store. J Biol chem. 277:9219-9225, 2002.
Richard SJ. Lithium and GSK-3: one inhibitor, two inhibitory actions, multiple outcomes. Pharmacological Sciences. 24:441-443, 2003.
Roderer G. On the toxic effects of tetraethyl lead and its derivatives on the chrysophyte Poterioochromonas malhamensis. VII. Protective action of thiol compounds, vitamins, trace elements and other agents. Ecotoxicology and Environmental Safety. 11:277-294, 1986.
Almeida S, Domingues A, Rodrigues L, Oliveira CR and Rego AC. FK506 prevents mitochondrial-dependent apoptotic cell death induced by 3-nitropionic acid in rat primary cortical cultures. Neurobiology of Diease. 17:425-444, 2004
Sakahira H, Enari M, Ohsawa Y, Uchiyama Y and Nagata S. Apoptotic nuclear morphological change without DNA fragmentation. Curr. Biol. 9：543-546, 1999.
Sastry PS, Rao KS. Apoptosis and the nervous system. Journal of Neurochemistry. 74(1):1-20, 2000.
Segal DS, Kuczenski R. Individual differences in responsiveness to single and repeated amphetamine administration: behavioral characteristics and neurochemical correlates. Journal of Pharmacology & Experimental Therapeutics. 242(3):917-26, 1987.
Shaw KP, Pu CE, Lin DL, Liu JC. and Fong JM. A comparative study of inhalation and self-administration methamphetamine-induced toxicities in rats. Advance Forensic Science. 5:264-273, 1995a.
Shaw KP, Pu CE, Chen L, Hu SH, and Fong JM. 50 Human Methamphetamine-related Victims. Epidemiological, Pathological and Toxicological Studies. Advance Forensic Science. 5:274-281, 1995b.
Sinclair AJ. Bayer AJ. Johnston J. Warner C. Maxwell SR. Altered plasma antioxidant status in subjects with Alzheimer's disease and vascular dementia. International Journal of Geriatric Psychiatry. 13(12):840-5, 1998.
Stephans SE, Yamamoto BK. Methamphetamine-induced neurotoxicity: roles for glutamate and dopamine efflux. Synapse. 17(3):203-9, 1994.
Stennicke GS, and Salvesen J. Biochemical characteristics of caspases-3, -6, -7, and -8: Biol. Chem. 272, 25719, 1997.
Stumm G, Schlegel J, Schafer T, Wurz C, Mennel HD, Krieg JC, and Vedder H. Amphetamine induce apoptosis and regulation of bcl-splice variants in neocortical neurons. FASEB Jouronal. 13(9):1065-1072, 1999.
Tewari M, Quan L, O’RourkeK, Desnoyers S, Zeng Z, Beidler, DR, Poirier GG, Salvesen GS, and Dixit VM. Yama/CPP32b, a mammalian homolog of ced-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell. 81：801–809, 1995.
Trump BF and Arstila AU. cell injury and cell death. In principles of pathobiology. pp.9-95, 1971.
Senatorov VV, Ren M, Kanai H, Wei H and Chuang D-M. Short-term lithium treatment promotes neuronal survival and proliferation in rat striatum infused with quinolinic acid, an excitotoxic model of Huntington’s disease. Molecular Psychiatry. 9:371-385, 2004.
Wang HG, Pathan N, Ethell IM, Krajewski S, Yamaguchi Y, Shibasaki F, McKeon F, Bobo T, Franke TF, Reed JC. Ca2+-induced apoptosis through calcineurin dephosphorylation of Bad. Science 284, 339– 343, 1999.
Wang JY, Shum AY, Ho YJ, and Wang JY. Oxidative neurotoxicity in rat cerebral cortex neurons: synergistic effects of H2O2 and NO on apoptosis involving activation of p38 mitogen-activated protein kinase and caspase-3. Journal of Neuroscience Research. 72(4):508-19, 2003.
Westerink BHC. The effects of drugs on dopamine biosynthesis and metabolism in the brain. Academic London. 224-291, 1979.
Wise RA. Catecholamine theories of reward: a critical review. Brain Research. 152(2):215-47, 1978.
Xiaohua Li, Terence A. Ketter, Mark A. Frye. Synaptic, intracellular, and neuroprotective mechanisms of anticonvulsants: are they relevant for the treatment and course of bipolar disorders. Journal of Affective Disorders. 69:1-14, 2002.
Xiaolin D, Ning SC, Michanel T, McCoy, Weiguo C, Michanel A, and Jean LC. Methamphetamine induces apoptosis in an immortalized rat striatal cell line by activating the mitochondrial cell death pathway. Neuropharm. 42：537-845, 2002.
Luo Y , Smith JV, Paramasivam V, Burdick A, Curry KJ and Butko P. Inhibition of amyloid-β aggregation and caspase-3 activation by the Ginkgo biloba extract EGb761. PNAS. 99:12197-12202, 2002.
Zbigniew K. Binienda. Neuroprotective Effects of L-Carnitine in Induced Mitrochondrial Dysfunction. Ann. N.Y. Acad. Sci. 993:289-295, 2003.
Zbigniew K. Binienda, Syed F. Ali. Neuroprotective role of L-Carnitine in the 3-nitropropionic acid induced neurotoxicity. 125:67-73, 2001.
Zhang J. Su Y. Oury TD. Piantadosi CA. Cerebral amino acid, norepinephrine and nitric oxide metabolism in CNS oxygen toxicity. Brain Research. 606(1):56-62, 1993.
Zhou L. J. and Zhu X. Z. Reactive oxygen species-induced apoptosis in PC12 cells and protective effect of bilobalide. J. Pharmacol. Exp. Ther. 293: 982–988, 2000.
Zsolt Bacso, and James F. Measurement of DNA damage associated with apoptosis by laser scanning cytometry. Cytometry. 45:180-186, 2001.

行政院衛生署管制藥品管理局：台灣地區緝獲毒品統計表，2002。
法務部統計處：法務統計摘要，pp1-18，1999。
徐人英：藥效藥劑學與臨床藥品動態學，合計圖書出版社，1982。