臺灣博碩士論文加值系統

1-甲基-4-酚基-1,2,3,6-四氫嘌呤（1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 簡稱MPTP）是一種具有選擇性之神經毒性物質。對黑質-紋狀體多巴胺神經傳導途徑有高度專一性的傷害，能引發與人類巴金森氏症相似的顫抖、肌肉僵直及起始運動困難等症狀。
本實驗的目的主要在探討MPTP導致鼠腦氧化壓力的情形。我們採用兩個月大及六個月大兩種年齡層的雄性ICR小白鼠，單次給予MPTP（30 mg/kg）注射於腹腔，控制組則只注射溶劑，做急性期（24小時以內）的觀察。在不同時間犧牲動物後，取出腦組織分成大腦額葉、次皮質區、紋狀體、中腦及小腦等區用以檢測。利用分光光譜儀（spectrophotometer）測量超氧陰離子（O2·-）之生成速率、測量總超氧物歧化脢（SOD）和過氧化氫脢（catalase）之活性，用高液態色層分析之螢光測定法（high performance liquid chromatography fluorescence detection）測定MDA-TBA 聚合物的量。比較二月齡和六月齡小白鼠的O2·-生成速率則是利用化學螢光法（chemiluminescence）監測。
經本論文實驗結果我們發現：小白鼠經MPTP處理後，在大腦額葉、次皮質區和小腦的粒線體產生O2·-的速率在四小時達最大值。鼠腦的總超氧物歧化脢活性和過氧化氫脢活性增加的時間先後不同。其中紋狀體和中腦總超氧物歧化脢活性增加持續至二十四小時，而過氧化氫脢活性則在第八小時達到最高。MDA含量有隨時間增加而增加的情形，至二十四小時達最大值。6月齡的小白鼠經MPTP處理後，中腦超氧陰離子（O2·-）的生成速率較大且丙二醛（MDA）含量較高，超氧物歧化脢（SOD）和過氧化氫脢（catalase）活性較低。可見經MPTP處理後6月齡鼠腦較2月齡鼠腦所遭受的氧化壓力有較大的趨勢。
總而言之，單次劑量的MPTP注射處理對小白鼠確實造成氧化壓力增加的情形，其中以紋狀體及中腦較為明顯；而MPTP所引起的氧化壓力在6月齡的小白鼠中腦比2月齡的小白鼠中腦更為明顯。

A selective neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is converted into 1-methyl-4-phenyl-2,3-dihydropyridium ion (MPDP+) by astrocytes. After autoxidation, the toxic metabolite 1-methyl-4-phenylpyridinium (MPP+) is formed and cause damage to the dopaminergic neurons. In the mouse, the MPTP-induced etiology similar to Parkinsonian syndromes has been extensively studied.
In this study, we investigated the MPTP-induced oxidative stress in the mouse brain. Two- and six-month old male ICR mice were used. At 1, 2, 4, 8, 10, 12 or 24 hr after single injection (i.p.) of MPTP (30 mg/kg), the mice were sacrificed. Brains were rapidly removed from the skull and selected areas (frontal cortex, subcortex, striatum, midbrain and cerebellum) dissected out and processed for various biochemical estimations. The time-dependent changes of mitochondrial O2.- generation rate, activities of superoxide dismutase (SOD) and catalase in the brain were measured by spectrophotometry. Lipid peroxidation (measured as MDA content) in the brain were also determined by high performance liquid chromatography fluorescence detection. The comparison of mitochondrial O2.- generation rate between 2- and 6-month old mice was made by chemiluminescence.
Results showed that in the frontal cortex, the subcortex and the cerebellum, the mitochondrial O2.- generation peaked at 4 hr after MPTP treatment. In the striatum and the midbrain, the activity of total SOD increased with time. However, the peaked activity of catalase was observed at 8 hr in the frontal cortex and the striatum and 10 hr in the midbrain. The MDA content also increased with time after MPTP injection. As compared with 2-month old, the 6-month mouse brain generated higher level of O2.- 4 hr after MPTP-treatment. They also had higher content of MDA. But the activities of SOD and catalase were lower in the midbrain of 6-month old mice after MPTP-administration.
Taken together, these results showed that a single dose of MPTP-injection increased oxidative stress in the mouse brain, particularly in the striatum and the midbrain. In 6-month old mice, the oxidative stress caused by MPTP was more prominent in the midbrain. On the basis of the present results, the oxidative stress is crucial in neurotoxicity caused by MPTP.

圖表目錄……………………………………………………………………III
中文摘要……………………………………………………………………V
英文摘要……………………………………………………………………VII
壹、緒論………………………………………………………………………1
巴金森氏症……………………………………………………………………1
MPTP…………………………………………………………………………2
MPTP與自由基的產生………………………………………………………3
抗氧化系統……………………………………………………………………4
氧化壓力………………………………………………………………………4
MPTP與氧化壓力……………………………………………………………5
實驗目的………………………………………………………………………5
貳、材料與方法………………………………………………………………7
一、實驗設計…………………………………………………………………7
（一）O2·-組…………………………………………………………………7
（二）SOD及catalase組………………………………………………………7
（三）MDA組………………………………………………………………7
（四）2月齡及6月齡比較組…………………………………………………7
二、動物組織…………………………………………………………………7
三、測量O2·-之生成速率……………………………………………………8
（一）分離粒線體……………………………………………………………8
（二）製備SMP………………………………………………………………8
（三）測量方法………………………………………………………………8
1、利用分光光譜儀（spectrophotometer）測量O2·-之生成速率…………8
2、利用化學螢光法（chemiluminescence）測定O2·-生成速率……………9
四、測量總超氧物歧化脢和過氧化氫脢之活性……………………………10
（一）測量總超氧物歧化脢（total SOD）之活性……………………………10
（二）測量過氧化氫脢的活性………………………………………………11
五、丙二醛（MDA）的測定…………………………………………………11
六、蛋白質含量的測定………………………………………………………12
七、統計方法…………………………………………………………………13
參、結果………………………………………………………………………14
一、O2·-的產生速率……………………………………………………………14
二、總超氧物歧化脢的活性變化……………………………………………14
三、過氧化氫脢的活性變化…………………………………………………15
四、MDA的含量情形…………………………………………………………15
五、2月齡和6月齡小白鼠受MPTP處理後各腦區四種參數的比較…………16
肆、討論………………………………………………………………………17
伍、結論………………………………………………………………………21
陸、參考文獻…………………………………………………………………22
柒、圖表………………………………………………………………………30
圖表目錄
表一 小白鼠經MPTP處理後各腦區在不同時間產生O2·-的速率……………30
表二 小白鼠經MPTP處理後各腦區不同時間總超氧物歧化脢活性變化……31
表三 小白鼠經MPTP處理後各腦區在不同時間過氧化氫脢的活性變化……32
表四 白鼠經MPTP處理後各腦區在不同時間MDA的含量情形………………33
表五 2月齡和6月齡小白鼠受MPTP注射處理後各腦區四種參數的比較……34
圖一 MPTP代謝圖………………………………………………………………35
圖二 MPTP於腦中代謝途徑之簡圖一…………………………………………36
圖三 MPTP於腦中代謝途徑之簡圖二…………………………………………37
圖四 氧自由基的生成與抗氧化系統對抗的作用模式圖………………………38
圖五 利用細胞色素C還原法間接檢測次粒線體物質產生O2·-的速率…………39
圖六 化學螢光法檢測O2·-生成速率的原理……………………………………40
圖七 分光光譜儀測定超氧物歧化脢的原理……………………………………41
圖八 過氧化氫脢活性的測定原理………………………………………………42
圖九 丙二醛（MDA）含量的測定原理…………………………………………43
圖十 在小白鼠的額葉、次皮質和小腦粒線體產生超氧自由基的情形………44
圖十一 MPTP注射四小時後對小白鼠大腦產生超氧自由基的影響……………45
圖十二 小白鼠額葉、紋狀體和中腦的超氧物歧化脢活性隨時間的變化圖……46
圖十三 小白鼠額葉、紋狀體和中腦的過氧化氫脢活性隨時間的變化圖………47
圖十四 MDA脂質過氧化物在注射MPTP後在三腦區增加的情形………………48
圖十五 2和6月齡小白鼠受MPTP注射後O2·-的產生速率、總超氧物歧化的活性、過氧化氫脢活性變化及MDA含量情形…………………………………………………………………………………49

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