臺灣博碩士論文加值系統

帕金森氏症為一種好發於中老年人的神經退化性疾病，致病型式可區分為遺傳型與環境毒物型，主要的臨床現象為運動失調、顫抖等不自主的症狀。之前文獻報導指出由病理學研究帕金森氏症的致病機轉，發現有神經損傷情形造成dopamine分泌下降而無法執行正常神經衝動，之後由生化研究更深入指出，粒線體complexΙ受到環境毒物如(MPTP、rotenone)等攻擊，使得ROS (reactive oxygen species)增加造成氧化壓力促使神經細胞apoptosis或degeneration。本論文中利用環境毒物rotenone與MPTP處理human dopaminergic neuroblastoma SH-SY5Y cells進行研究，探討當細胞受到毒物攻擊模擬帕金森氏症的致病過程中，抗氧化物質對神經保護的能力。從MTT assay、trypan blue exclusion assay等實驗方法，發現rotenone、MPTP可以毒殺神經細胞，計算rotenone的IC50大於1 μM，MPTP的IC50介於1 mM到2 mM中，而且在從PI stain、DNA fragmentation等實驗證明，細胞死亡的機制是透過apoptosis，處理0.5 μM rotenone的分佈於 sbu-G0/G1的細胞約佔27%，處理1 mM MPTP時分佈sub-G0/G1的細胞約佔36%。此外由 dihydroethidium (DHE) 偵測細胞中superoxide的含量，發現ROS可以被誘導上升，推論ROS的上升與細胞走向apoptosis有關。為了確認上述現象，細胞合併處理BSO以降低細胞中glutathion的含量，發現rotenone、MPTP的致死情形更為明顯，顯示ROS增加與神經細胞傷害有關。而根據先前文獻指出帕金森氏症的指標性蛋白α-synuclein會在神經細胞內堆積，而造成α-synuclein增加的原因推論是由ROS攻擊正常的α-synuclein而產生不正常堆疊所造成。在本論文的實驗結果發現處理rotenone或MPTP培養24小時會促使α-synuclein的量增加，顯示rotenone及MPTP可以誘導類似帕金森氏症生成的症狀。以western blotting進一步證明促使apoptosis的機制是透過活化caspase 3、PARP。而在合併處理抗氧化劑的實驗，證明抗氧化劑（baicalein、N-actylcystein、Acetyl -L-carnitine、lipoic acid）可以減緩rotenone、MPTP處理24小時所促使的細胞凋亡，而且caspase 3、PARP的活化明顯受到抑制，推論抗氧化劑是透過清除ROS減低caspase的活化，達到保護神經細胞的功能。

Recently, it have been shown that rotenone and MPTP, a specific inhibitor of mitochondrial complexⅠ, is a useful tool in animal model of Parkinson’s disease. In this thesis, a human dopaminergic neuronblastoma SH-SY5Y cell line was used to study the effect of exposure to rotenone and MPTP on cell death, activation of stress-induce pathway and aggregation ofα-synuclein, the characteristic protein accumulated in lewy bodies and mimic the pathogenesis of Parkinson’s disease. Moreover it was investigated that the role of antioxidants play in the rotenone- and MPTP-induced α-synuclein aggregattion, oxidative stress and apoptosis. The results showed that both toxins induce a time and dose-dependant decrease in cell survival with IC50 approached values of 0.5 μM after 48hr for rotenone and 1 mM after 24h for MPTP, respectively. Rotenone and MPTP induced apoptosis was demonstrated by using trypan blue exclusion assay, PI staining, DNA fragmentation, Western blotting assay. In addition, it was found that rotenone and MPTP induced apoptosis an increase in ROS production. The Rotenone- and MPTP-induced cell death and caspase 3 activation were reduced by pretreatment with antioxidants (N-acetyl-cystein (10 mM), Baicalein(40 μM), Acetyl-L-Carnitine(0.5 mM) and Lipoic acid (0.1 mM)),indicated a role of ROS in the neurotoxicity. These results suggested that the antioxidants prevent neurons cells from rotenone and MPTP induce apoptosis through decreasing oxidative stress.

目錄：
縮寫表 4
中文摘要 6
英文摘要 8
緒論 9
實驗 一、試劑 25
二、儀器 28
三、方法 30
1.細胞培養 30
2.細胞存活率試驗(MTT Assay) 30
3.Trypan blue exclusion assay 31
4.觀察細胞外形 32
5.細胞週期分佈分析(PI stain) 32
6.DNA fragmentation assay 33
7.利用流式細胞儀分析細胞內的活性氧分子 34
8.caspase 3 activity assay 35
9.western blotting 35
結果 38
1. Rotenone可以毒殺neuroblastoma SH-SY5Y細胞 38
2.MPTP可以毒殺neuroblastoma SH-SY5Y cells 39
3.Rotenone的細胞毒性是透過誘導細胞凋亡 40
4.MPTP的毒性是透過誘導 SH-SY5Y行細胞凋亡 41
5.Rotenone、MPTP會增加活性氧分子生成的增加 41
6.Rotenone、MPTP會活化PARP與Caspase 3 42 7.Rotenone and MPTP 會促使α-synuclein的堆積 43
8.合併處理抗氧化劑可以減緩rotenone、MPTP所造成的細胞毒性 44
9.抗氧化物可以降抵rotenone、MPTP所誘導的活性氧分子的生成 46
10.抗氧化物可以降低由rotenone、MPTP所促使caspase 3與
PARP的活化 47
11.MPTP處理1mM有活化JNK的現象 48
討論 59
參考文獻 66
表圖 78

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