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研究生:黃雅妮
研究生(外文):Ya-Ni Huang
論文名稱:第一型血紅素氧化酶於甲基安非他命神經毒性之角色
論文名稱(外文):The Role of Heme Oxygenase-1 on Methamphetamine-induced Neurotoxicity
指導教授:王家儀王家儀引用關係
指導教授(外文):Jia-Yi Wang
學位類別:博士
校院名稱:國防醫學院
系所名稱:生命科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:98
語文別:中文
論文頁數:95
中文關鍵詞:甲基安非他命維生素C第一型血紅素氧化酶神經毒性p38有絲分裂-活化蛋白質激酶神經細胞-神經膠質細胞共同培養
外文關鍵詞:MethamphetamineVitamin CHeme oxygenase-1Neurotoxicityp38 mitogen-activated protein kinaseNeuro/glia coculture
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甲基安非他命(methamphetamine, METH),安非他命的衍生物,為常見的濫用藥物。臨床及動物研究顯示,甲基安非他命具強效的神經毒性。文獻亦證明,甲基安非他命之神經毒性不單是單胺類神經細胞,亦造成不同的腦區神經細胞損傷。如甲基安非他命誘導皮質神經細胞毒性(neurotoxicity)產生,導致認知(cognitive)及運動(motor)功能受損。第一型血紅素氧化酶(heme oxygenase-1, HO-1),又稱為壓力性蛋白(stress protein),已被證實具細胞保護的功能以對抗不同的神經損傷。相反地,研究發現過度血紅素氧化酶活性導致過量游離鐵離子釋放,將導致神經毒性。然而,甲基安非他命誘導皮質神經細胞毒性及第一型血紅素氧化酶表現之相關性,目前仍不清楚。
本論文第一部分實驗,以大腦皮質神經細胞/膠質細胞培養(cortical neuron/glia cocultures)之模式,探討甲基安非他命是否誘導第一型血紅素氧化酶表現及第一型血紅素氧化酶於甲基安非他命之神經毒性之角色為何。細胞給予不同濃度的甲基安非他命處理,隨著甲基安非他命濃度增高,細胞毒性愈明顯。細胞給予5 mM甲基安非他命處理,誘導50%的神經細胞死亡及伴隨著膠質細胞活化的現象。即時螢光定量反轉錄-聚合酶連鎖反應及西方墨點法證明,細胞給予甲基安非他命處理後顯著地誘導細胞內第一型血紅素氧化酶mRNA及蛋白質的表現,其誘導在細胞毒性產生之前。雙重及三重螢光免疫染色證明,甲基安非他命誘導第一型血紅素氧化酶表現在活化的星狀膠質細胞(astrocyte)、微膠質細胞(microglia)及活的神經細胞(viable neurons),而非死亡的神經細胞(dying neurons)。抑制p38有絲分裂-活化蛋白質激酶訊息傳遞路徑(p38 mitogen-activated protein kinase pathway),顯著阻斷甲基安非他命誘導第一型血紅素氧化酶的表現及加重其神經毒性。細胞給予血紅素氧化酶抑制劑tin protoporphyrine IX (SnPP),顯著地減少血紅素氧化酶的活性且加重甲基安非他命之神經毒性。然而,細胞給予cobalt protoporphyrine IX (CoPP)處理先誘導第一型血紅素氧化酶表現具有部分保護神經細胞免於甲基安非他命的毒性。此部分的結果證明,甲基安非他命經由p38有絲分裂-活化蛋白質激酶訊息傳遞路徑誘導第一型血紅素氧化酶表現產生神經保護作用,但不足以完全保護神經細胞對抗甲基安非他命毒性。因此,著重於第一型血紅素氧化酶做為藥物治療的觀念,已廣泛地被接受。
本論文第二部分實驗,仍以大腦皮質神經細胞/膠質細胞培養之模式,探討維生素C是否能誘導第一型血紅素表現及維生素C減少甲基安非他誘毒性是否藉由第一型血紅素氧化酶之機制。維生素C已被證實具有改善甲基安非他命誘導之神經毒性之作用,然而其作用機制仍不清楚。我們以大腦皮質神經細胞/膠質細胞培養之模式,探討維生素C是否藉由誘導細胞表現第一型血紅素氧化酶之機制,改善甲基安非他命之毒性。培養之神經細胞/膠質細胞給予維生素C (1, 3, 5 mM)處理後結果發現,隨著維生素C的濃度越高及處理時間增長,細胞內第一型血紅素氧化酶基因及蛋白質的表現隨之增加。此外,細胞給予p38有絲分裂-活化蛋白質激酶抑制劑(SB203580)有效抑制維生素C誘導細胞第一型血紅素氧化酶基因及蛋白表現;然而給予ERK訊息傳遞路徑抑制劑則無此效果,即證明維生素C藉由p38有絲分裂-活化蛋白質激酶訊息便遞路徑誘導第一型血紅素氧化酶之表現。細胞先給予維生素C處理30分鐘再給予甲基安非他命,結果發現其第一型血紅素氧化酶基因及蛋白的表現量顯著高於單獨給予甲基安非他命處理的細胞。同時也發現,細胞給予維生素C處理後,可明顯地抑制甲基安非他命誘導產生的自由基及改善甲基安非他命誘導的細胞毒性。然而,細胞給予第一型血紅素氧化酶活性抑制劑(SnPP),則有效地阻斷維生素C對抗甲基安非他命誘導之自由基生成及神經保護作用。實驗結果證明,維生素C可能藉由p38有絲分裂-活化蛋白質激酶之訊息傳遞路徑,誘導細胞表現第一型血紅素氧化酶參與對抗甲基安非他命毒性之神經保護作用機制。因此,維生素C誘導第一型血紅素氧化酶之機制可作為預防或改善甲基安非他命之神經毒性之應用。
Methamphetamine (METH), a methyl derivative of amphetamine, is a drug of abuse worldwide. METH can cause neuropsychiatric and neurotoxic damage in humans and animals. Despite the prominent toxic effect in monoaminergic neurons, METH also has deleterious effects in widespread brain regions. The impairment of cognitive and motor functions caused by METH administration underscores the importance of METH toxicity in cortical neurons. Heme oxygenase-1 (HO-1), a stress protein, exerts an important cellular cytoprotective mechanism against various neuronal injuries. In contrast, excessive HO activity and subsequent release of free iron result in neurotoxicity. However, the possible involvement of HO-1 in the protection or exacerbation in METH-induced toxicity in cortical neurons is unclear.
The first part of this thesis focused on the role of HO-1 induction by METH neurotoxicity in cortical neuron/glia cocultures. Exposure of cultured cells to various concentrations of METH led to cytotoxicity in a concentration-dependent manner. The METH concentration of 5 mM, which caused 50% of neuronal death with accompanied glial activation, was chosen for subsequent experiments. RT-PCR and Western blot analysis revealed that METH significantly induced HO-1 mRNA and protein expression, both preceded cell death. Double and triple immunofluorescence staining further identified HO-1-positive cells as activated astrocytes, microglia, and viable neurons, but not dying neurons. Inhibition of the p38 mitogen-activated protein kinase (MAPK) pathway significantly blocked HO-1 induction by METH and aggravated METH neurotoxicity. Inhibition of HO activity using tin protoporphyrine IX (SnPP) significantly reduced HO activity and exacerbated METH neurotoxicity. However, prior induction of HO-1 using cobalt protoporphyrine IX partially protected neurons from METH toxicity. Taken together, our results suggest that induction of HO-1 by METH via the p38 MAPK signaling pathway may be protective, albeit insufficient to completely protect cortical neurons from METH toxicity. Targeting HO-1 to achieve therapeutic benefits, like in many diseases, may have a potential to prevent METH toxicity.
The second part of this thesis focused on the induction of HO-1 expression by vitamin C (Vit. C) on METH neurotoxicity in cortical neuron/glia cocultures. Vit. C has been reported to attenuate METH-induced neurotoxicity in rats by attenuating stiatal dopamine and serotonin depletions. However, whether Vit. C has an effect on HO-I induction to attenuate METH-induced neurotoxicity remains unclear. Using primary cortical neuron/glia cocultures, we explored the effect of Vit. C on the expression of HO-1 in METH toxicity. Cultured cells incubated with Vit. C (1,3, 5 mM) resulted in concentration-and time-dependent induction of HO-1 mRNA and protein. The HO-1 induction by Vit. C was blocked by SB203580 (a p38 MAPK inhibitor) but not by PD98059 (an ERK MAPK inhibitor). Pre-treatment with Vit. C (5 mM) at 30 min prior to METH exposure significantly elevated the levels of HO-1 mRNA and protein as well as attenuated ROS production and METH neurotoxicity. However, inhibition of HO activity using SnPP abolished both effects of attenuation by Vit. C on METH-induced ROS production and neurotoxicity. Taken together, these data suggest that Vit. C enhances HO-1 expression via p38 MAPK pathway and contributes, at least in part, to alleviate METH neurotoxicity. We suggest that HO-1 induction by Vit. C may serve as a strategy to alleviate METH neurotoxicity.
正文目錄 ----------------------------------------------------------------------- I
圖目錄 ----------------------------------------------------------------------- II
縮寫全名對照表 ----------------------------------------------------------------------- V
中文摘要 ------------------------------------------------------------------------ VIII
英文摘要 ----------------------------------------------------------------------- XI

緒論 ----------------------------------------------------------------------- 1
實驗目的 ----------------------------------------------------------------------- 20
材料與方法 ----------------------------------------------------------------------- 22
實驗結果
第一部分 ----------------------------------------------------------------------- 32
第二部分 ----------------------------------------------------------------------- 48
討論
第一部分 ----------------------------------------------------------------------- 41
第二部分 ----------------------------------------------------------------------- 54
結論
第一部分 ----------------------------------------------------------------------- 47
第二部分 ----------------------------------------------------------------------- 58
總結與未來展望 ----------------------------------------------------------------------- 59
參考文獻 ----------------------------------------------------------------------- 62
附錄 ----------------------------------------------------------------------- 95
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