臺灣博碩士論文加值系統

　　近年來流行病學報導，罹癌風險與環境風險因子，例如：廚房油煙、二手菸與奈米粒子有很大的相關性，然而，關鍵在於找出環境風險因子引發癌症風險的生物指標物，最廣為人知的致癌物以多環芳香烴化合物為例，PAHs活化的代謝路徑透過PAH-o-quinones，其會導致ROS生成，會與生物結構反應，例如：蛋白質、脂質與DNA，其加成物可以用來判別個體或群體是否暴露於罹癌、職業風險或環境化學物質中。因此本研究利用全面性代謝質體學，探討評估環境風險因子對於人體健康的影響，稱之為環境代謝質體學。
　　本研究之研究目的為鑑定代謝輪廓與BaP誘導發生肺腫瘤、新穎性組蛋白去乙醯酶HDACI對肺癌小鼠之間的關係性，以液相層析質譜分析法建立代謝質體學(metabolomics)為平台的分析技術，結合主成分分析(principal component analysis：PCA)，利用液相層析儀串聯三段式質譜儀偵測注射BaP之小鼠與注射HDACI小鼠尿液與血清中胺基酸與核苷的含量變化。
　　注射BaP十週後，小鼠血清中的脯胺酸(Pro)、纈胺酸(Val) 、甲硫胺酸(Met)、穀氨醯胺(Gln)與組胺酸(His)隨BaP劑量上升而下降之趨勢，核苷則是腺苷(A)、N2-甲基鳥苷(N2-mG)與去氧腺苷(dA)則隨BaP劑量上升而上升之趨勢，另外，注射HDACI於種植肺癌細胞之小鼠血清中的苯丙胺酸(Phe)、甘胺酸(Gly)、丙胺酸(Ala)、脯胺酸(Pro)、甲硫胺酸(Met)與穀胺酸(Glu)為顯著性下降，核苷則是黃核苷(X)與去氧腺苷(dA)顯著性下降。因此本研究認為胺基酸與核苷能夠做為早期發現暴露於BaP與注射HDACI之生物指標物。

　　Epidemiological studied have investigated that cancer risk in the worldwide is associated with exposure to several environmental risk factors such as cooking oil fumes (COF), environmental tobacco smokes (ETS) and nanoparticles. However, it is of crucialto determine biomarkers to promote exposure and cancer risk assessments related to environmental risk factors. Especially, PAHs are best known for their carcinogenicity, several metabolic pathways of PAHs activation through PAH-o-quinones, leads to ROS generation; react with many biological structures including protein, lipids and DNA. These adducts can be used to identify individuals and populations at risk for developing cancer and also for setting human exposure limits for occupational and environmental chemicals. Our utilization of metabolomics is to globally understand the assessment of environmental risk to human health in a new frontier termed “environmental metabolomics”.
　　The objective of this study was to classify any changes in metabolite profiles associated with the development of BaP-induced lung tumors and treated HDACI in mice models. Using a metabolomicsstrategy linking a liquid chromatography – mass spectrometry-based approach in conjunction with principalcomponent analysis and confirmation by liquid chromatography triple quadrupole tandem mass spectrometry, we havedemonstrated that the amino acids profiles and nucleosids eprofiles of the urine andserum of BaP-treated mice and HDACI-treated mice are changed.
　　Five amino acids(Pro, Val, Met,Gln and His)were significantly reduced and three nucleosides(A, N2-mG and dA) also increasedin serum of BaP-treated mice at10weeks after treatment.In the other hand, sixamino acids(Phe, Gly, Ala, Pro, Met and Glu) were significantly reduced and twonucleosides(A and N2-mG) also decreased in serum of HDACItreated mice at 6 weeks after treatment.
　　Our results suggest that amino acids and nucleosidesprofiles may be useful as an early indicator of the presence ofBaP-induced lung tumors and HDACItreated mice.

Abstract III
目錄 V
圖目錄 IX
表目錄 XIII
附錄表目錄 XVI
附錄圖目錄 XVIII
第一章　緒論 1
1.1　研究背景 1
1.2　組蛋白去乙醯酶 3
1.3　苯(a)芘 6
1.4　8-OHdG 8
1.5　代謝質體學概要 9
1.5.1　代謝質體學的研究策略及流程 11
1.5.2　代謝質體學之相關研究 15
1.6　藥物代謝 17
1.7　液相層析儀串聯質譜儀原理及構造 18
1.7.1　液相層析原理 18
1.7.2　質譜儀構造與原理 18
1.8　研究目的 23
1.8.1　苯(a)芘 23
1.8.2　組蛋白去乙醯酶抑制劑 24
第二章　研究方法 25
2.1　實驗架構 25
2.2　實驗藥品 26
2.3　儀器及設備 28
2.4　動物實驗 29
2.4.1　腹腔注射BaP組 29
2.4.2　腹腔注射HDACI於種植肺癌細胞之小鼠 34
2.5　LC-Q-TOF 37
2.5.1　血清樣品前處理 37
2.5.2　 LC-Q-TOF 分析方法 37
2.6　主成分分析(PCA) 40
2.7　LC-MS/MS定量樣品前處理 41
2.7.1　尿液及血清樣品中之胺基酸定量樣品前處理 41
2.7.2　尿液及血清樣品中之核酸定量樣品前處理 43
2.8　LC-MS/MS分析方法 45
2.8.1　胺基酸定量分析方法 45
2.8.2　核苷定量分析方法 47
2.8.3　 8-OHdG定量分析方法 50
第三章　結果與討論 53
Part I注射苯(a)芘於表皮生長因子基因突變之小鼠 53
3.1　LC-Q-TOF分析結果 53
3.2　主成分分析結果 60
3.2.1　正離子模式 60
3.2.2　負離子模式 75
3.3　表皮生長因子基因突變小鼠肺腫瘤發生率 89
3.4　方法確效 91
3.4.1　胺基酸 91
3.4.2　胺基酸方法確效 92
3.4.3　核苷 96
3.4.4　核苷方法確效 97
3.5　血清與尿液中胺基酸與核苷定量分析結果 105
3.5.1　基因型不同之差異 105
3.5.2　暴露於BaP之影響 106
3.5.3　BaP誘發腫瘤生物指標物 111
3.5.4　產生腫瘤與否之差異性 115
3.5.5　小鼠飼養時間長短之差異性 122
3.6　尿液樣品 125
3.6.1 　胺基酸 125
3.6.2　核苷 128
3.7　 8-OHdG定量分析結果 131
Part II注射HDACI於種植肺癌細胞之小鼠 133
3.8　尿液與血清中胺基酸定量分析 133
3.9　尿液與血清中核苷定量分析 137
3.10　 8-OHdG定量分析結果 139
3.11　不同品系小鼠內生性胺基酸濃度 140
第四章結論 141
第五章　參考文獻 164

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