臺灣博碩士論文加值系統

背景：細胞色素P450 (cytochrome P 450，CYP)為在人體中負責許多物質氧化代謝的酵素，包括致癌物質等。而CYP2A6基因為CYP家族中一個重要的成員，已有許多研究報導CYP2A6與癌症的發生率有顯著的相關性。口腔癌是臺灣死亡率相當高的癌症之一，為了達到早期發現的目的，急需方便而快速經濟的篩檢方式
高解析熔點(High Resolution Melting , HRM)是利用緩慢提高溫度使DNA互相分離，具有突變的異型接合子DNA解離熔點不同於正常或同型結合子，激發出螢光值不同進而可分辨其差異。
材料與方法：在本研究中，從正常人60位及口腔癌患者55位之周邊血液萃取其DNA，並對於CYP2A6基因以HRM進行分析，此為第一次利用HRM之技術來分析口腔癌患者CYP2A6基因的變異。
結果：發現存在有基因型CYP2A6*/ CYP2A6*9相對於CYP2A6*1A/ CYP2A6*1A者對於口腔癌發生率較低。並且偵測到基因型CYP2A6*1A/CYP2A6*1A， CYP2A6*1A/CYP2A6*1B，CYP2A6*1A/CYP2A6*5，CYP2A6*1A/CYP2A6*7， CYP2A6*1A/CYP2A6*9，CYP2A6*1B/CYP2A6*1B， CYP2A6*1B/CYP2A6*5，CYP2A6*1B/CYP2A6*7，CYP2A6*1B/CYP2A6*9，CYP2A6*5/CYP2A6*9，CYP2A6*/ CYP2A6*9。
結論：將可提供一個新而快速篩檢口腔癌可能發生族群的模式，使之於在口腔癌的預防及治療上更具有潛力。

ACKGROUND:CYP is now widely known as a family of enzymes metabolizing a wide variety of xenobiotics, including drugs and carcinogens. Human cytochrome P450 2A6(CYP2A6) is an important member of the CYPs and CYP2A6 has been reported that associated with risk of cancer
Oral squamous cell carcinoma(OSCC) is one of the high lethality in Taiwan, to discover the cancer appear at the early year, we extremely need a fast, convenient and cheap way to screen OSCC.
High Resolution Melting Analysis(HRMA) detect and distinguish wild type homozygote, heterozygote and mutative homozygote by fluorescence differ at the melting temperature .

Material and Method:DNA sample from peripheral blood of 60 normal men and 55 OSCC patient/men, analysis the genotype and allele frequency by Q-PCR and HRMA. This is the first time to to analyze the CYP2A6 polymorphism by HRMA.

Results:Genotype of CYP2A6, CYP2A6*1A/CYP2A6*1A, CYP2A6*1A/CYP2A6*1B, CYP2A6*1A/CYP2A6*5, CYP2A6*1A/CYP2A6*7, CYP2A6*1A/CYP2A6*9, CYP2A6*1B/CYP2A6*1B, CYP2A6*1B/CYP2A6*5, CYP2A6*1B/CYP2A6*7, CYP2A6*1B/CYP2A6*9, CYP2A6*5/CYP2A6*9, CYP2A6*9/CYP2A6*9, and allele of CYP2A6 were deteced.
The frequency with which the subjects carried homozygotes of the CYP2A6* 9 which causes lack of the enzyme activity, was lower in the OSCC patients than in the healthy control subjects. The odds ratio (OR) of the group homozygous for the CYP2A6* 9 was significantly lower and calculated to be 0.08 (95% CI; 0.01–0.77) when the OR for the population with homozygotes ofthe CYP2A6 wild-type gene was defined as 1.00. In the allelic-base analysis, there was also a significant decrease in the OR for the CYP2A6*9 allele.

Conclusion: A new and fast way to detect the group with high risk of OSCC and we suggest that CYP2A6* 9/ CYP2A6* 9 play an important role to decrease the risk of OSCC

致謝 i
中文摘要 1
Abstract 3
第一章：緒論 5
第二章：文獻回顧 7
第一節：抽煙與口腔癌之流行病學 7
第二節：口腔癌引發因子 9
第三節：細胞色素P450 2A6 11
第四節：高溶解曲線分析High Resolution Melting Analysis(HRMA) 14
第三章：材料與方法 16
第一節：實驗材料 16
血液DNA萃取 16
巢式聚合酶連鎖反應(Nest-PCR) 16
即時定量聚合酶連鎖反應(real-time quantitative PCR, Q-PCR)以及高溶解曲線分析 17
基因定序 17
第二節：實驗流程 18
第三節：實驗方法 19
實驗檢體 19
萃取血液DNA 19
引子設計Primer design 20
巢式PCR(nest-PCR) 21
及時定量PCR(real-time quantitative PCR)與高溶解曲線分析(High Resolution Melting Analyzsis ) 23
基因定序 24
DNA定序(需要避光) 25
統計分析 26
第四章：研究結果 27
第一節：依臨床資料來分類 27
第二節：以HRM檢視CYP2A6各外顯子之SNP 29
第三節：CYP2A6基因型與與口腔鱗狀上皮細胞癌的相關性分析 31
第四節：CYP2A6對偶基因型(allele)與與口腔鱗狀上皮細胞癌的相關性分析 33
第五章：討論 34
第一節：高溶解曲線分析與其他測定單點核苷酸突變(SNPs)技術比較 34
第二節：高溶解曲線分析的限制 37
第三節：CYP2A6各型與癌症之關連性 39
第六章 未來展望 42
第七章：結論 43
第八章：參考文獻 44
第表九章：圖次 50
附圖一、肝臟中phaseI及phaseII代謝反應所參與酵素。 50
附圖二、Roche LightCycler® II 480 51
圖一、巢式PCR第一次反應完成後2％ Agarose gel做電泳分析確定產物。 52
圖二、第一外顯子以HRM方法顯示之基因分群 53
圖四、第二外顯子以HRM方式 顯示基因分群 55
圖五、第五外顯子以HRM方式 顯示基因分群 56
圖六、第五外顯子定序後發現之SNP位置 57
圖七、第六外顯子以HRM方式 顯示基因分群 58
圖八、第六外顯子定序後發現之SNP位置 59
圖九、第七外顯子以HRM方式 顯示基因分群 60
圖十、第七外顯子定序後發現之SNP位置 61
圖十一、第八外顯子定序後發現之SNP位置 62
圖十二、第八外顯子及內含子變異以HRM方式 顯示基因分群 63
圖十三、第九外顯子第一部份定序後發現之SNP位置 64
圖十四、第九外顯子第一部份變異以HRM方式顯示基因分群 65
圖十五、第九外顯子第二部份變異以HRM方式顯示基因分群 66
圖十六、以引子偵測多個不連鎖基因的方式 67
表一、在各地區CYP2A6之allele frequency 68
表二、本研究使用之引子 69
表三、研究對象之基本資料 70
表四、口腔鱗狀細胞癌病人臨床基本資料 71
表六、CYP2A6基因型於口腔鱗狀上皮細胞癌病人與健康對照組中之分佈情況 73
表七、CYP2A6基因型於口腔鱗狀上皮細胞癌病人與健康對照組中之分佈情況 74
表八、CYP2A6各基因型存與否在於口腔鱗狀上皮細胞癌病人與健康對照組中之分佈情況： 75

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