臺灣環境中的鉛(lead)暴露被認為可影響認知、學習和記憶障礙，更可能危害人及動物並導致疾病，甚至死亡的重金屬之ㄧ。過去流行病學研究指出，環境中的重金屬鉛為導致阿茲海默症(Alzheimer’s disease; AD)可能的危險因子。慢性鉛暴露同時伴隨著發生細胞壞死(necrosis)，細胞程式凋亡(apoptosis)以及類澱粉蛋白沉積(β-amyloid deposition)。在神經細胞中β-amyloid形式主要藉由細胞自噬(autophagy)及內噬作用(endocytosis)降解胞器中富含類澱粉先驅蛋白(amyloid precursor protein; APP)而得。因此，本研究目的從新觀點探討在鉛暴露(lead exposure)下的類澱粉蛋白生成異常(amyloidogensis disorders)中自噬作用可能扮演的角色。以人類神經母細胞瘤細胞株SH-SY5Y細胞分化模式，添加10 μM 視網酸(retinoic acid; RA)誘導細胞分化模式1-4天將細胞分化成不同階段的神經細胞形態。利用MTT assay分析未分化及分化神經細胞在5 μΜ醋酸鉛處理後之細胞存活率，以TUNEL分析偵測斷裂的DNA以評估細胞凋亡情形。同時藉由即時定量聚合酶鏈鎖反應及酵素免疫分析法分別定量APP與類澱粉蛋白Aβ1-40與類澱粉蛋白Aβ1-42片段表現量。並以西方點墨法觀察細胞自噬作用標幟蛋白LC3II及自噬作用訊息分子mammalian target of rapamycin (mTOR)蛋白磷酸化變化，以評估可能的訊號傳遞調控路徑。結果發現，未分化及分化的神經細胞SH-SY5Y在鉛暴露下都會造成LC3I轉變成LC3II增強細胞自噬作用，無論有無誘導分化的SH-SY5Y神經細胞均造成細胞之程式性死亡。同時，伴隨著自噬效應增強，mTOR的磷酸化現象也會有減弱趨勢。另外，比較未分化神經細胞與經過RA分化後神經細胞對於鉛造成的毒性，結果顯示未分化神經細胞對鉛毒性耐受性較差，可以佐證前臨床研究，鉛對胎兒與嬰幼兒傷害較為嚴重。

Lead (Pb) is one of the most well known toxic heavy metals in human beings and animals, which leads to toxic neurological disorders, cognitive problems, learning and memory disabilities. Epidemiological studies revealed that chronic lead exposure is one of the environmental risk factors which may cause Alzheimer’s Disease, which were speculated for the observation of cellular necrosis, apoptosis, and β-amyloid deposition frequently occuring altogether after chronic lead exposure. Recent studies have shown that the β-amyloid formed during autophagic turnover of APP-rich organelles supplied by both autophagy and endocytosis. Therefore, we will conduct the new perspective for studying the possible role of autophagy on amyloidogensis disorders after lead exposure. SH-SY5Y human neuroblastoma cells, used in this study, were differentiated to a neuronal phenotype by retinoic acid (RA) to the culture medium at 10 μM for 1, 2, 3 and 4 days. Doses of lead acetate with of lead acetate were 5 μM and applied to the neuronal culture and then cell viability measurement by MTT assay. The apoptotic effect of non-differentiation and differentiation neuroblastoma cells after lead exposure was determined by cleaved DNA fragments. Furthermore, APP, intracellular Aβ1-40 and Aβ1-42 expression were quantified by Real-time PCR and ELISA, respectively. The autophagy process and variation of total and phosphorylated mammalian target of rapamycin (mTOR) forms were determined after lead exposure in non-differentiation and differentiation neuroblastoma cells by western blot. The results indicate that lead exposure enhances autophagy response in both non-differentiation and differentiation SH-SY5Y cells, which might cause neuronal apoptosis associated with β-amyloidgenesis. Otherwise, lead exposure resulted in the inhibition of mTOR signaling, which correlated with the autophagic process. Besides, in our studies, non-differentiated cells exhibited more toxic vulnerability than RA induced differentiated neuron is congruous to previous finding that lead exposure during fetal development might be a potential risk factor for AD in the adulthood.

中文摘要 ... IV
英文摘要 ... VI
英文名詞縮寫對照表 ... VIII

第一章 前言與文獻回顧 ... 1

1.1 鉛與阿茲海默病... 1
1.2 類澱粉蛋白與阿茲海默病 ... 2
1.3 γ-secretase activity在形成類澱粉蛋白中的角色 ... 6
1.4自噬作用在類澱粉蛋白形成與沉積所擔任的角色 ... 7
1.5 Mammalian target of rapamycin (mTOR) signaling pathway與自主吞噬調控 ... 12
1.6類澱粉蛋白與自噬作用實驗之神經細胞分化模式 (Differentiation on neuronal cell model) ... 14

第二章 研究動機與目的 ... 16

第三章 實驗架構 ... 18

第四章 研究材料與方法 ... 19

4.1 細胞培養(Cell culture) ... 19
4.2 免疫螢光染色法(Immunofluorescence) ... 20
4.3 細胞毒性之分析(MTT assay) ... 21
4.4 細胞程式凋亡分析(Apoptosis assay) ... 22
4.5 酵素免疫分析(ELISA assay) ... 23
4.6 抽取total RNA(RNA extraction) ... 24
4.7反轉錄 cDNA聚合酵素連鎖反應(Reverse transcription reaction, RT) ... 24
4.8即時定量聚合酶連鎖反應(Real-time Polymerase Chain Reaction, Q-PCR) ... 25
4.9 西方墨點法(Western blotting) ... 26
4.1 統計方法(Statistical method) ... 30

第五章 研究結果 ... 31

5.1 利用Retinoic acid 促進神經細胞分化成具有dopaminergic phenotype功能成熟神經胞 ... 31
5.2 醋酸鉛暴露在未分化以及分化狀態下神經細胞存活率與細胞凋亡變化 ... 31
5.3 醋酸鉛暴露與APP、β-Amyloid [1-40]及β-Amyloid [1-42]表現量影響 ... 32
5.4 醋酸鉛暴露給予壓力後未分化以及分化神經細胞與自噬效應與mTOR kinase變化 ... 34

第六章 研究討論與結論 ... 36

第七章 未來研究方向 ... 45

第八章 參考文獻 ... 47

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