臺灣博碩士論文加值系統

本論文以小鼠神經母細胞瘤Neuro-2a細胞株為實驗對象，探討螯合劑與銅的複合物(PDTC-Cu+2 complex)以及環境污染物-氯化鎘和甲基汞(MeHg)造成神經母細胞瘤細胞進行程式性凋亡的訊息傳遞路徑。
鎘為環境中常見的污染物。在動物實驗模式中，鎘為高度神經致毒物。我們發現氯化鎘(CdCl2)可以引起細胞核凝集的形態改變，凋亡體的形成以及hypodiploid DNA等現象，並且可以引起單細胞的彗星圖騰，因而推測氯化鎘引起細胞死亡是經由細胞凋亡的途徑。進一步研究顯示Neuro-2a 細胞在處理100mM的氯化鎘後，在五分鐘後偵測到細胞內鈣離子濃度大幅上揚。另外，我們也發現細胞內自由基的濃度增加，粒線體膜電位降低，在增加氧化性壓力後，伴隨呈現出抑癌基因p53的表現增加。已知p53的增加，會促使BAX gene表現，因此我們使用Western blotting技術發現pro-apoptotic protein BAX，亦被偵測到其表現隨氯化鎘處理有增加的現象。p38的表現也有增加且MAPK 抑制劑PD98059及p38抑制劑SB203580皆可抑制細胞凋亡。因此，我們根據以上結果推論鈣離子的上升和氧化性壓力的增加，在氯化鎘毒性機轉中扮演決定性的角色，並經由p53和p38等途徑進而引起細胞凋亡。除此以外，鈣離子活化calpain 所引起的細胞凋亡亦參與其中。
PDTC具抗氧化性及螯合金屬的雙重作用，不但廣用於農業殺菌劑及殺黴劑，並作為臨床上銅及鎳之中毒之解毒劑。目前此類化合物被研究發展成抗癌及抗愛滋病的藥物。銅是生理必須微量元素，我們實驗室發現PDTC與銅的複合物會高度的增強彼此的毒性作用，而其他金屬如鐵、鋅、鉛則無此增強作用。本論文的研究結果，也發現PDTC-Cu2+複合物引起神經母細胞瘤的毒性極強，而銅離子的專一性螯合劑BCPS可以完全的抑制此毒性。另外，利用ICP-MS偵測到複合物處理的細胞，細胞內含銅量會大量增加，而BCPS亦可以抑制此現象，故推測此複合物引起的細胞毒性與細胞內銅含量增加有關。PDTC-Cu2+複合物在Neuro-2a細胞引發細胞核凝集的形態改變，凋亡體的形成以及hypodiploid DNA等現象，並且可以引起單細胞的彗星圖騰，證實複合物引起細胞死亡是經由細胞凋亡的途徑。
接著我們觀察到PDTC-Cu2+複合物會造成Neuro-2a細胞內自由基的濃度增加，粒線體膜電位降低。而且發現到抑癌基因p53和pro-apoptotic protein BAX有顯著增加。p38的表現也有顯著增加且MAPK 抑制劑PD98059及p38抑制劑SB203580皆可抑制細胞死亡。因此，我們由這些結果推論鈣離子的上升和氧化性壓力的增加，在PDTC-Cu2+複合物毒性機轉中扮演決定性的角色，並經由p53和p38等途徑進而引起細胞死亡。除此以外，鈣離子活化calpain 所引起的細胞凋亡亦參與其中。
中樞神經系統為甲基汞主要的目標器官，會造成中樞神經系統中毒，導致神經退化性疾病。而我們研究的結果顯示Neuro-2a細胞處理30mM甲基汞後，會造成細胞內鈣離子濃度上升，進一步研究顯示，甲基汞會降低粒線體膜電位，增加活性氧化物質生成，在增加氧化性壓力後甲基汞可以持續增加p53、p38和BAX的表現。因此甲基汞引發Neuro-2a細胞凋亡的訊息傳遞途徑是[Ca+2]i增加 ® ROS增加 ® 粒線體功能異常 ® 惡性循環促使[Ca+2]i 及ROS更增加® DNA damage ® p53增加 ® BAX增加 ® 細胞凋亡。
綜合本論文的研究結果，我們發現這三種金屬化合物的共通性質是均能促使Neuro-2a細胞內鈣及ROS增加，繼而粒線體功能異常，DNA受損，p53增加 最後導致BAX增加而引起細胞凋亡。除此主要作用途徑外，其他途徑如calpain 活化也應考慮在內。

The aim of this paper is focused on the exploration of the mechanism of cytotoxicity induced by the PDTC-Cu+2 complex, environmental pollutants-CdCl2 and MeHg in the cultured Neuro-2a cells.
Cadmium is a heavy metal with high toxicity and animal studies have shown that cadmium is a potent neurotoxicant. That the changes of morphology including nuclear condensation, apoptotic body formation and hypodiploidity of DNA suggested that CdCl2 induced cell death through an apoptotic process in the cultured Neuro-2a cells. We also provided evidence that CdCl2 exerted comet pattern on single cell in the cultured Neuro-2a cells. In addition, the intracellular concentration of calcium [Ca+2]i rapidly rise followed by the decreased the mitochondrial membrane potential and increased reactive oxidative species production. The increased oxidative stress subquently increased the expression of the tumor suppressor gene p53 BAX proteins. In addition, CdCl2 caused an increase expression of p38 and PD98059 and SB203580 could protect cytotoxicity induced by CdCl2. Therefore, we conclude from these findings that the death signaling pathway of CdCl2 is mediated by the increase of intracellular calcium and oxidative stress production which then trigger the subsequent p53 and p38 signaling pathway. Another possibility is that the cytotoxicity effects of CdCl2 were due to Ca+2-activated protease, calpain.
The chelating and antioxidant effects of pyrrolidine dithiocarbamate (PDTC) have been extensively investigated for preventing cell death induced by different insults. Cu+2 is an essential metal in our body. In our laboratory, we found that PDTC and Cu+2 in the complex form markly potentiated with each other. By contrast, other metals (Fe+3, Zn+2, and Pb+2) had no such potentiating effects on PDTC. In this study, we showed that the PDTC-Cu+2 is very potent in inducing apoptotic process in the cultures Neuro-2a cells; morphological nuclear condensation and apoptoic bodies formation changes and were apparently due to the increase of intracellular Cu contents. The non-complex the non- permeable copper-specific chelator BCPS could prevent the cytotoxicity. Thus, we concluded that the increase of intracellular Cu content is crucial for the complex in inducing apoptosis in the cultured Neuro-2a cells. Studuies on the signaling pathway showed that the intracellular concentration of calcium [Ca+2]i rapidly rise. Further studies showed that the complex decreased the mitochondrial membrane potential and increased reactive oxidative species production. Following the increased oxidative stress, the complex induced the increase expression of the tumor suppressor gene p53 and BAX protein. In addition, the complex caused an increase expression of p38 and PD98059 and SB203580 could protect cytotoxicity induced by the complex. Therefore, we concluded that the death signaling pathway of the complex was mediated by the increase of intracellular calcium and oxidative stress production which subsequently triggered the p53 expression and p38 signaling pathway. Another possibility is that the cytotoxic effects of the complex were due to Ca+2-activated protease, calpain.
MeHg is a neurotoxin in the central nervous system. In this study, we found that MeHg caused an increase of intracellular calcium concentration [Ca+2]i. We also found that MeHg significantly decreased mitochondrial membrane potential ΔYm and increased ROS generation. The downstream signaling pathway of elevated ROS in the cultured Neuro-2a cells were subsequently investigated. Our results showed that MeHg caused an increase of p53, p38 and BAX, which preceded the onset of apoptosis. Thus, the death signaling pathway of MeHg in the cultured Neuro-2a cells includes [Ca+2]i increase ® ROS generation ® DNA damage ® p53 and BAX expression ® apoptosis.
In conclusion, the death signaling pathways induced by CdCl2, PDTC-Cu+2 complex and MeHg are summarized on Fig. 33.

Abbreviations……………………………………………………..1
Abstract in Chinese…………………………………………….…2
Abstract in English……………………………………………..…5
Introduction…………………………………………………….…9
Materials and methods…………………………………………...17
Results……………………………………………………………27
Discussion………………………………………………………..47
References……………………………………………………….55
Figures and tables………………………………………………..62

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