臺灣博碩士論文加值系統

帕金森氏症(Parkinson’s disease)是常見的一種神經退化性疾病。帕金森氏症病患有顫抖、身體僵直、運動遲緩等現象，並且無法自主行動，主要的病理特徵是位於人類中腦黑質緻密區中多巴胺神經元逐漸死亡。過去研究顯示氧化壓力的形成與多巴胺神經元的缺失有關聯性。多巴胺為神經傳導分子，很容易被氧化造成細胞內氧化壓力增加。另外，在帕金森氏症病患的腦部病理組織切片發現位於黑質緻密區的鋅離子含量有明顯增加的情形。本實驗室之前的研究顯示鋅離子與多巴胺造成的神經細胞死亡具有協同作用，並且在大鼠腦部紋狀體區域造成多巴胺減少的現象，但相關的分子機制仍未知。在本研究中我建立了一個初代大鼠中腦細胞培養模式系統。利用此模式發現鋅離子以及多巴胺共同處理時對於中腦神經細胞的死亡具有協同作用，同時造成中腦神經細胞的樹突產生不連續呈現點狀。在短時間內以低濃度的鋅離子以及多巴胺處理下，粒線體的移動速度以及數量有顯著減少，顯示粒線體的運輸受到損傷，可能為導致細胞死亡的原因之一。此外，神經元細胞核有不正常的萎縮，TUNEL染色結果顯示有將近一半的非多巴胺神經元會走向細胞凋亡，但多巴胺神經元並不會進行細胞凋亡。兩種抗氧化劑，乙酰半胱胺酸 (N-acetylcysteine, NAC)及麩胱甘肽(glutathione, GSH)，對於鋅離子與多巴胺造成多巴胺神經元死亡具有保護作用，因而推測氧化壓力為鋅離子與多巴胺造成神經細胞死亡的主要原因之一。利用本研究所建立的大鼠中腦初代神經細胞可以確認之前以PC12細胞受鋅離子與多巴胺作用所造成的死亡為氧化壓力所造成的結果，結果顯示多巴胺神經元並不會進行細胞凋亡，以及粒線體的移動可能與神經細胞退化相關。

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. The main pathological hallmark of PD is pronounced loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and depletion of striatal dopamine (DA). However, the molecular mechanisms responsible for the loss of DA neurons remain unknown. Our previous study has shown that Zn2+ synergistically enhances dopamine-induced apoptotic cell death in PC12 cells, and cause DA depletion in the rat striatum, suggesting the roles of Zn2+/DA in the pathogenesis of PD. To examine the possible involvement of Zn2+/DA in the death of DA neurons in PD and its mechanisms, I established a primary rat mesencephalic culture model in this study. My results showed that Zn2+ combined with DA treatment caused a synergistic effect on the death of cultured rat mesencephalic cells and damage to neuronal processes. The mitochondrial transportation along the processes was inhibited upon Zn2+/DA treatment. The cell death induced by treatment of Zn2+ and DA is partially mediated by apoptosis in non-DA neurons but apoptosis was not found in DA-neurons. In addition, antioxidants, N-acetylcysteine (NAC) and glutathione (GSH), are able to rescue cells from the cell death induced by Zn2+/DA, suggesting that oxidative stress may be the main cause of neuronal death. My results from this study not only confirm the previous results obtained by PC12 cells but provide a further understanding of the mechanisms involved in the Zn2+/DA toxicity.

Table of Contents
中文摘要i
Abstract ii
Abbreviations iii
Table of Contents v
I. Introduction 1
1. Parkinson’s disease 1
2. Nigrostriatal dopaminergic pathway 1
3. Abnormal protein aggregation 2
4. Oxidative stress 2
5. Mitochondrial dysfunction 3
6. Dopamine 4
7. Zinc 5
8. Apoptosis：Type I cell death 6
9. Autophagy：Type II cell death 7
10. Necrosis：Type III cell death 8
II. Specific Aims 9
III. Materials and Methods 10
1. Materials 10
2. Primary cultures of rat mesencephalic neurons 10
3. Immunofluorescent staining (IF) 11
4. TUNEL assay 11
5. Time-lapse confocal imaging 12
6. Statistics 12
IV. Results 13
1. Establishment of a model to study the synergistic death by Zn2+ and DA 13
2. Zn2+ and DA caused synergistic death of cultured rat mesencephalic cells 13
3. The effects of Zn2+ and DA on the mitochondria transportation in the cultured rat mesencephalic cells 15
4. Cell death induced by treatment of Zn2+ and DA is partially mediated by apoptosis in non-DA neurons but not in DA-neurons 15
5. Antioxidants NAC and GSH inhibited the Zn2+- and DA-induced neurotoxicity in the cultured rat mesencephalic cells 16
V. Discussion 18
References 22
Figures 31
Fig. 1 Identification of rat embryonic mesencephalic neurons in DIV15. 31
Fig. 2 Glial cells in the rat mesencephalic culture at DIV15. 32
Fig. 3 DA neurons in the rat mesencephalic culture at DIV15. 33
Fig. 4 The effects of Zn2+ and DA on the induction of cell death in the cultured rat mesencephalic culture. 34
Fig. 5 The effects of Zn2+ and DA on the induction of DA neurons death in the cultured rat mesencephalic culture. 36
Fig. 6 The effects of Zn2+ and DA on the mitochondria transportation in the cultured rat mesencephalic cells. 38
Fig. 7 Zn2+/DA- induced death of non-DA neurons is partially mediated by apoptosis. 40
Fig. 8 The death pathways of DA neurons induced by Zn2+/DA are not mediated by apoptosis. 42
Fig. 9 The protective effect of NAC on the Zn2+- and DA-induced neurotoxicity. 44
Fig. 10 The protective effect of GSH on the Zn2+- and DA-induced neurotoxicity. 47
Appendix 1. Stages in the dissection of the ventral mesencephalon from the 14 day rat embryos. (Dunnetta and Björklundb, 1997). 50
Appendix 2. 51

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