在受傷的腦或神經疾病中可以見到活化的微小膠細胞，且微小膠細胞可能會在受傷的腦和其他的腦疾病中分泌特殊的神經因子。帕金森氏症是一種神經退化性疾病。雖然目前致病的原因不是很清楚，但氧化壓力可能是導致這類疾病的主要原因。先前的研究顯示多巴胺神經元可能會因為多巴胺長時間或高濃度的作用而造成細胞的死亡。也有證據顯示帕金森氏症病人死後腦部黑質體的鋅離子濃度比一般人來得高，而且鋅離子會進入細胞而達到毒害的影響。本論文的研究顯示鋅離子會加強多巴胺或過氧化氫所導致的PC12細胞死亡；而這樣的結果可以被人類微小膠細胞的條件培養液所抑制。條件培養液經過熱處理後仍然具有保護作用，證明了條件培養液含有熱穩定的因子。實驗中也發現條件培養液的特異性：PC12細胞在條件培養液的環境作用下可以抑制A23187和C2-ceramide所造成的細胞死亡，卻無法抑制staurosporine的毒殺作用。本論文也發現在多巴胺和鋅離子的作用下所導致的PC12細胞死亡過程中會引發JNK的磷酸化；而在條件培養液作用下，JNK磷酸化的程度變少了，顯示條件培養液的保護作用抑制了JNK的死亡路徑。因此，由本論文的結果顯示，微小膠細胞會分泌一些因子以幫助細胞抵抗死亡而保護神經元。

Microglia have the potential to produce specific neurotrophic molecules in response to injury and brain diseases. Activated microglia are seen after brain injury or in neurological disease, such as Parkinson’s disease (PD). PD is a progressive neurodegenerative disorder. Although its cause remains unknown, it is believed that enhanced oxidative stress is a major component in the pathogenesis of nigral cell death in PD. Previous results have shown that DA induces apoptosis of dopaminergic neurons in a time- and concentration- dependent manner. In addition, a number of studies have shown that Zn2+ may enter the cell to reach toxic concentrations and that Zn2+ concentration is higher in the striatum of the postmortem brains of PD patients than that of the control brains. We have previously shown that Zn2+ synergistically enhanced the dopamine- and H2O2- induced PC12 cell death. To study the role of microglia in the cell death, I have examined the effect of conditioned medium from a human microglia cell line on the PC12 cell death induced by dopamine and Zn2+. The result shows that conditioned medium inhibits the PC12 cell death and the phosphorylation of JNK induced by dopamine and Zn2+ is diminished by the conditioned medium. It appears that the factor(s) that are responsible for the protection is heat-stable because the conditioned medium heated in 70℃for 30 minutes still has the ability to protect the cell death. Cell death induced by A23187 and C2-ceramide, but not by staurosporine can be protected by the conditioned medium. Results from this study suggest that the microglia secrete some factors which can protect neuron.

中文摘要 iv
Abstract v
縮寫表 vi
緒論 1
帕金森氏症(Parkinson’s disease, PD) 1
帕金森氏症的致病機轉與治療 2
神經退化中的氧化壓力 3
鋅離子與帕金森氏症 4
微小膠細胞 (microglia cell) 條件培養液 (conditioned medium) 的保護作用 6
研究動機與目的 7
實驗材料與方法 8
一、材料 8
二、細胞培養 8
三、條件培養液的製作 9
四、培養盤覆蓋poly-L-lysine 9
五、多巴胺與鋅離子的細胞毒性實驗 10
六、細胞存活率分析 10
七、統計分析方法 11
八、聚丙醯膠體電泳 (SDS-PAGE) 與西方墨點法 (Western Blotting) 11
1. 樣品收集 11
2. 定量蛋白質濃度 11
3. SDS-PAGE 11
4. 轉漬 12
5. 免疫染漬（immunoblotting） 12
九、條件培養液的特異性 12
結果 13
一、多巴胺與鋅離子對PC12細胞死亡的影響 13
二、鋅離子加強了過氧化氫 (H2O2) 的毒性 13
三、條件培養液的保護作用 14
四、條件培養液的保護能力 14
1. 熱處理後的條件培養液 14
2. 稀釋後的條件培養液 15
3. 藥物毒害時條件培養液的保護作用 15
五、不含血清培養液與條件保養液中，MAPK (mitogen-activated protein kinase) 之表現比較 15
討論 18
參考文獻 23
圖一、鋅離子和多巴胺對PC12細胞死亡的影響。 31
圖二.、鋅離子作用下加強了過氧化氫 (H2O2) 的毒性。 32
圖三、微小膠細胞的條件培養液保護PC12細胞抵抗鋅離子和多巴胺及過氧化氫所造成的影響。 33
圖四、熱處理過的條件培養液仍然具有保護作用。 34
圖五、稀釋後條件培養液仍舊可以幫助PC12抵抗鋅離子和多巴胺所造成的細胞死亡。 35
圖六、PC12在條件培養液環境下其他藥物的影響。 36
圖七、PC12細胞內的JNK和ERK磷酸化程度的表現。 37
附錄 38
附錄一、帕金森氏症病人腦中的黑質體退化。 38
附錄二、腦部運動神經網絡與多巴胺。 39
附錄三. 活性氧與神經退化性疾病的發病機制。 41

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