臺灣博碩士論文加值系統

Tetrachlorohydroquinone（TCHQ）四氯對苯二酚為Pentachlorophenol（PCP）五氯酚的主要毒性代謝產物之一；PCP被廣泛使用於木材防腐劑及生物殺菌劑上，為目前重要的環境污染物之一。PCP的主要代謝產物TCHQ已知會造成細胞之DNA單股斷裂(single-strand breaks, SSB)，而具有基因毒性及細胞毒性，且TCHQ亦被證實其產生的氧化性壓力及毒性均較PCP來得高。因此推測PCP致癌的能力來自於TCHQ的毒性所致。
在前趨的動物二階段致癌實驗中證實，PCP誘發及促進腫瘤產生的能力，高於TCHQ，推測這可能是因為，TCHQ會導致細胞走上細胞凋亡(apoptosis)的能力高於PCP，因而降低了TCHQ致癌的能力。本研究會對TCHQ 是否可誘發細胞進行細胞凋亡及其作用機轉作進一步的研究。
研究中發現NIH/3T3纖維母細胞在TCHQ8小時50μM劑量的作用下，有明顯細胞凋亡的現象；而在一些控制細胞凋亡的重要基因中發現Bcl-2蛋白的表現量有上升的趨勢，並且會隨著時間的增加而降低；p53蛋白及cytochrome c蛋白的表現，隨著時間的增加有上昇的趨勢；此外亦發現隨著劑量及時間的增加，caspase 9與caspase 3的活性亦有增加的趨勢，另外在3T3 over-expression Bcl-2細胞株中發現其caspase 9與caspase 3並無上升的趨勢，證實了TCHQ造成3T3細胞凋亡是經由mitochondria-mediated的路徑。
在抗氧化劑方面篩檢出captopril、penicillamine、dihydrolipoic acid (DHLA)及2,3-dimercaptosuccinic acid (DMSA)會抑制TCHQ的細胞毒性及基因毒性，而這些化合物皆屬於thiol group；另一方面發現lipoic acid(LA)雖然無法抑制TCHQ的毒性但其還原態的化合物DHLA卻可以保護3T3細胞免於TCHQ的毒性，顯示這些抗氧化劑的保護作用可能來自於-SH group可以與ROS作用而降低其對DNA的傷害所致。

Tetrachlorohydroquinone (TCHQ) has been identified as a major toxic metabolite of the widely used wood preservative pentachlorophenol and also implicated in its genotoxicity.
In our preliminary two-stage carcinogenesis experiment, it was proved that the tumor promotion activity of PCP was higher than TCHQ but the toxicity of TCHQ was higher than PCP. We assumed that TCHQ, but not PCP, may cause apoptotic cell death and thus decrease the tumor promotion activity and tumorigenecity. This study was designed to test the hypothesis that TCHQ induced apoptotic cell death in 3T3 cells and to investigate the mechanisms of apoptosis induced by TCHQ. Moreover, we identified some antioxidants with the ability to reduce TCHQ-induced ROS, and defined whether these agents may inhibit cytotoxicity and genotoxicity induced by TCHQ.
In the present study, we found that apoptotic cell death can be induced in 3T3 cells treated with 50μM TCHQ for 8 hrs. The generation of ROS and collapse of mitochodrial transmembrane potrntial has been observed to occur in early apoptosis induced by TCHQ, and the process is related to bcl-2 protein expression. The activity of caspase9 and caspase3 subsequently increased with a time- and dose- dependent pattern. Bcl-2 over-expressed 3T3 cells provided marked protection against the cytotoxicity induced by TCHQ. This result suggested that Bcl-2 protein play an important role in the process of apoptosis in 3T3 cells, which also further indicated that apoptotic cell death in 3T3 cells occurs through the mitochondria-mediated pathway.
Captopril, penicillamine, dihydrolipoic acid (DHLA) and 2,3-dimercaptosuccinic acid (DMSA) are antioxidants with thiol groups. All of them were found to counteract the TCHQ-induced cytotoxicity. No protective ability was found in lipoic acid, oxidized form of DHLA, indicated that the protection comes from the –SH group which could bond with ROS and reduces the ROS-mediated DNA damage. This study also demonstrates that the comet assay is a feasible method for measuring DNA damage induced by TCHQ. Using this method, we found that captopril provided marked protection against genotocixity induced by TCHQ.

第一章 序論
第一節 研究動機1

第二章 文獻探討
第一節 五氯酚2
第二節 四氯對苯二酚2
第三節 活性氧物種3
第四節 細胞凋亡4
4.1.1 p 53蛋白5
4.1.2 Bcl-2蛋白及Bax蛋白6
4.1.3 cytochrome c、caspase 9、caspase 36
第五節、抗氧化劑
5.1.1 Lipoic acid (LA)，dihydrolipoic acid（DHLA）7
5.1.2 Captopril.8
5.1.3 penicillamine.8
5.1.4 meso-2,3-dimercaptosuccinic acid（DMSA）8

第三章 研究目的9

第四章 研究材料與方法
第一節 研究材料10
第二節 研究方法
細胞培養13
細胞氧化性壓力偵測分析13
DNA萃取及瓊膠電泳偵測細胞凋亡14
細胞存活率偵測分析（MTT assay）15
細胞基因毒性偵測分析（Comet assay）15
Mitochondrial transmembrane potential之偵測分析17
西方墨點法18
Caspase 活性分析.21

第五章 實驗架構.22

第六章 實驗結果
第一節、Tetrachlorohydroquinone（TCHQ）造成3T3細胞形態上的改變23
第二節、TCHQ引起3T3細胞凋亡23
第三節、 TCHQ造成3T3細胞內氧化性壓力增加24
第四節、抗氧化劑抵抗TCHQ對3T3細胞造成的細胞毒性25
第五節、抗氧化劑抵抗TCHQ對3T3細胞造成的基因毒性25
第六節、TCHQ造成3T3細胞mitochondria膜電位不穩定..27
第七節、Bcl-2蛋白在TCHQ造成細胞凋亡過程中的重要性27
第八節、3T3細胞內與細胞凋亡有關基因的表現28

第七章 討論
第一節、 TCHQ對NIH/3T3細胞之細胞凋亡機轉30
第二節、抗氧化劑抵禦TCHQ所造成細胞毒性及基因毒性31

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