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研究生:鍾育婷
研究生(外文):Yu-Ting Chung
論文名稱:Sulfotransferase 1A1單體型對臺灣男性族群口腔鱗狀上皮細胞癌易感性之影響
論文名稱(外文):Sulfotransferase 1A1 Haplotypes Associated with Oral Squamous Cell Carcinoma Susceptibility in Male Taiwanese
指導教授:劉宗榮劉宗榮引用關係
指導教授(外文):Tsung-Yun Liu
學位類別:博士
校院名稱:國立陽明大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:119
中文關鍵詞:磺基轉移酶1A1口腔鱗狀上皮細胞癌單體型液相層析串聯四極桿飛行時間質譜儀黃樟素檳榔嚼塊
外文關鍵詞:SULT1A1oral squamous cell carcinomahaplotypeLC/QTOF-MSsafrolebetel quid
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文獻指出無論是口腔癌患者的口腔組織,或是正常人的血液白血球,都能偵測到黃樟素DNA鍵結物。進一步分析個體環境因子的暴露情形,結果顯示在這些研究對象體內所偵測到黃樟素DNA鍵結物的形成,與嚼食檳榔的習慣具高度相關性。由於sulfotransferase是黃樟素DNA鍵結物形成的關鍵酵素,因此,本篇研究挑選160位口腔鱗狀上皮細胞癌男性患者,以及218位經年齡與性別相配對且無癌症病史者作為對照組,以序列分析的方式,探討SULT1A1編碼區之單體型對個體罹患口腔鱗狀上皮細胞癌之影響,進一步利用重組基因技術,分析基因多型性對sulfotransferase酵素活性的影響。結果顯示,445T與507T對偶基因,會顯著地提升罹患口腔鱗狀上皮細胞癌之風險。根據基因型分析結果,進一步挑選C445T (His149Tyr)、C507T、G600C與G638A (Arg213His)等多型性位點作為tagSNPs,以GENECOUNTING軟體分析SULT1A1編碼區(coding region)之單體型,結果顯示在同樣具有嚼食檳榔與吸菸習慣的族群裡,與單體型為haplotype a (野生型)者相比,若單體型屬於含有445T、507T與600C等對偶基因之haplotype c,罹患口腔鱗狀上皮細胞癌之風險顯著地升高(OR, 5.96; 95% CI, 1.60-26.15)。調整年齡、吸菸及嚼食檳榔等影響因子後,以haplotype a為基準進行複邏輯迴歸分析,結果顯示haplotype c與罹患口腔鱗狀上皮細胞癌的風險仍具顯著相關性(OR, 3.24; 95% CI, 1.57-6.68)。然而含有已知對酵素活性有顯著影響的G638A (SULT1A1213His)之haplotype b,對罹患口腔鱗狀上皮細胞癌風險的影響並不顯著。基因多型性對sulfotransferase酵素活性的影響方面,首先以2-naphthol為受質,將SULT1A1213Arg (638G)的活性視為基準,評估SULT1A1 allozyme在BL21(DE3)菌株中的活性,結果顯示SULT1A1149Tyr (445T)與SULT1A1213His (638A)的活性分別降低51%與72%。此外,以1'-hydroxysafrole為受質測試SULT1A1 allozyme之活性方面,先將由鼷鼠肝臟微粒體所催化形成的safrole-dGMP,利用液相串聯離子阱質譜儀及液相串聯四極桿飛行時間質譜儀加以鑑定,結果顯示合成之safrole-dGMP具有兩種異構物,分別為N2-(trans-isosafrol-3'-yl) deoxyguanosine 及 N2-(safrol-1'-yl) deoxyguanosine。再以由液相串聯質譜儀鑑定之safrole-dGMP作為標準品,評估SULT1A1 allozyme催化黃樟素的最終代謝物-1'-hydroxysafrole之sulfonation能力。結果顯示SULT1A1149Tyr (445T)與SULT1A1213His (638A)的活性分別降低33%與51%。綜論上述結果,本篇研究藉由單體型分析,提供另一種探討SULT1A1基因型對個體罹患口腔鱗狀上皮細胞癌之影響的評估方式,結果顯示SULT1A1基因多型性所造成酵素活性的差異,會改變人體代謝活化環境化學致癌物質的結果,進而影響個體對口腔鱗狀上皮細胞癌之易感受性。此外,對臺灣口腔癌盛行率影響極深的危險因子—嚼食檳榔的習慣,與SULT1A1單體型之間的交互作用,會顯著地修飾個體罹患口腔鱗狀上皮細胞癌之風險。
Previous studies demonstrated that safrole-DNA adducts are present in oral squamous cell carcinoma (OSCC) tissues and in human peripheral blood. The presence of safrole-DNA adducts is highly associated with personal betel quid chewing history. It’s well known that sulfotransferase is essential for the formation of safrole-DNA adducts. To elucidate the effects of SULT1A1 haplotypes on OSCC susceptibility, 160 male OSCC cases and 218 age- and sex-matched controls were screened for single nucleotide polymorphisms within the coding region of SULT1A1 by sequencing. The C445T and C507T polymorphisms were significantly associated with increased risk of OSCC. Based on the genotype analysis, haplotypes were constructed for C445T, C507T, G600C and G638A using GENECOUNTING software. The risk of OSCC was significantly increased in subjects who have betel quid chewing and cigarette smoking habits with haplotype c containing 445T, 507T, or 600C but not 638A (OR, 5.96; 95% CI, 1.60-26.15) as compared to the haplotype a (wild-type) carriers with the same habits. After adjustment for age, cigarette smoking and betel quid chewing, the haplotype c was significantly associated with increased risk of OSCC (OR, 3.24; 95% CI, 1.57-6.68) when compared to the haplotype a. The activity in sulfonation of 2-naphthol of recombinant His149Tyr (C445T) and Arg213His (G638A) variants was reduced by 51% and 72%, respectively when compared to that of the wild-type. Furthermore, the safrole-dGMP was synthesized in the presence of 1'-hydroxysafrole and mice liver microsomes. The synthesized safrole-dGMPs were characterized as N2-(trans-isosafrol-3'-yl) deoxyguanosine and N2-(safrol-1'-yl) deoxyguanosine by LC/ESI-ITMSn and LC/QTOF-MS. The LC/MS confirmed safrole-dGMP was used as reference standard to analyze the activity in sulfonation of 1'-hydroxysafrole of recombinant His149Tyr (C445T) and Arg213His (G638A) variants, which led to 33% and 54% reduced activity, respectively when compared to that of the wild-type. Taken together, haplotype analysis provides a novel evaluation of the SULT1A1 gene as a risk modifier on environmental carcinogen in OSCC and the association of SULT1A1 haplotypes with the risk of OSCC might be modified by betel quid chewing.
目錄.......................................................I
中文摘要..................................................II
英文摘要..................................................IV
第一章 背景介紹............................................1
第二章 研究動機與目的.....................................13
第三章 實驗材料與方法.....................................16
第四章 實驗結果...........................................31
第五章 討論...............................................41
第六章 結論...............................................51
參考文獻..................................................52
圖表......................................................60
附錄......................................................89
發表文章..................................................98
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