臺灣博碩士論文加值系統

本研究探討單碳代謝生化分子 (葉酸、B12及同半胱胺酸)與亞甲基四氫葉酸還原酶 ( methylenetetrahydrofolate reductase, MTHFR ) 第677 位點基因多型性與 (Ⅰ)肝腫瘤進展之相關性及(Ⅱ)血液淋巴球粒線體DNA損傷與肝癌罹患危險率之影響。以病例對照研究方法，自台南奇美醫院永康院區胃腸肝膽科門診收取初次診斷罹患肝癌病患90名 (病例組)，與性別年齡配對的正常健康對照組90名。檢測血清單碳代謝分子 (葉酸、B12與同半胱胺酸) 濃度，肝功能損傷指標GOT、GPT，與肝癌腫瘤指標AFP；分離血液單核淋巴球，以微生物法分析淋巴球內葉酸含量；抽取血液單核淋巴球總DNA，以即時定量聚合酶連鎖反應 ( real-time PCR ) 測定粒線體DNA斷損量與拷貝數，並針對MTHFR C677T單點突變設計引子與探針，作基因型分型觀察。研究結果(Ⅰ)顯示病例組60% 處於邊緣性葉酸缺乏營養狀態 ( 血清葉酸濃度 < 14 nM)，血清維生素B12濃度與肝腫瘤指標AFP濃度呈顯著正相關( r = 0.2657, P = 0.0119 )。血清葉酸濃度與肝腫瘤體積及肝腫瘤數目呈顯著負相關( r = -0.2227, P = 0.0349；r = -0.2742, P = 0.0089 )。以TNM分組定義病例組腫瘤進化參數，在T4與StageⅣ組血清葉酸濃度最低，B12與GOT濃度最高。複邏輯式回歸以血清葉酸營養狀態作腫瘤大小與數目以及肝癌腫瘤惡化危險率預測，結果發現邊緣性葉酸缺乏( 血清葉酸濃度 < 14 nM ) 可能增加腫瘤直徑大於5公分或多腫瘤顆粒數之風險 ( OR = 4.422, 95%C.I. = 1.272-15.373； OR = 6.375, 95%C.I. = 1.117-36.391 )。調整AFP濃度，葉酸濃度對腫瘤大小的預測力消失，對腫瘤數目之預測力仍存在。邊緣性葉酸缺乏(血清葉酸濃度 < 14 nM ) 亦可能增加腫瘤參數進展風險( T2以上OR = 6.148, 95%C.I. = 1.105-34.202；Stage Ⅱ以上OR = 6.375, 95%C.I. = 1.117-36.391 )，調整AFP濃度並不影響此趨勢。MTHFR C677T基因型分布比例無顯著差異。研究結果顯示(Ⅱ)病例組淋巴球粒線體DNA斷損量顯著高於對照組。與淋巴球及血清葉酸濃度呈顯著負相關，與血清維生素B12濃度、GOT與AFP濃度呈顯著正相關。複邏輯式回歸以粒線體DNA變異量及單碳代謝分子濃度預測肝癌罹患危險率，高粒線體DNA斷損量組( ΔCt≧ 5.32 ) 顯著增加5倍肝癌罹患危險率 (OR = 5.384, 95%C.I.= 1.71 -16.87)。高粒線體基因斷損量合併葉酸缺乏營養狀態，對肝癌罹患危險率顯著上升13倍 (OR = 13.62, 95% C.I. = 1.453-122.85)，調整GOT濃度後趨勢性消失。相較於677CC型，677TT型族群其血清葉酸濃度顯著下降，同半胱胺酸濃度顯著上升，病例組平均粒線體DNA斷損量顯著低於CC型與CT型 (P=0.0369)，且TT基因型在低血清葉酸濃度與高粒線體DNA斷損族群對肝癌罹患危險率具保護性 (OR = 0.016, 95% C.I. = 0.001-0.344; OR = 0.013, 95% C.I. = 0.001-0.539)。低淋巴球葉酸濃度與高血清維生素B12濃度下TT基因型對肝癌的罹患具保護性 (OR= 0.007, 95% C.I. = 0.001-0.097; OR = 0.023, 95% C.I. = 0.001-0.608)。綜合上述肝癌患者普遍處於葉酸缺乏營養狀態，血清葉酸濃度與B12濃度可預測肝腫瘤惡化危險率。肝癌患者淋巴球有粒線體DNA大片段斷損累積，可用以預測肝癌罹患危險率，高粒線體基因斷損量合併葉酸缺乏營養狀態，對肝癌罹患危險率顯著上升13倍。肝癌組TT型族群淋巴球粒線體DNA斷損顯著降低。高淋巴球粒線體DNA斷損族群具MTHFR 677TT基因型者其肝癌罹患危險率降低。顯示677TT型對粒線體DNA完整性及肝癌罹患率具保護性，機制未明，還需進一步的實驗討論。

The aims of the study are to investigate the relationships between one-carbon factors (folate, B12, homocysteine and MTHFR C677T polymorphisms) and tumor progression of hepatocellular carcinoma HCC ( I ) and mitochondrial DNA (mtDNA) large deletion (ΔmtDNA4.9kb) in peripheral blood lymphocytes (PBLs) and cancer risk of HCC (Ⅱ). In this case-control study, 90 HCC cases who were first time diagnosed as HCC patients, and 90 age- and sex- paired healthy controls were recruited from Chi-Mei Medcal Center. Blood samples were collected for the analysis of serum folate, B12, homocysteine levels, glutamicoxaloacetic transaminase (GOT), glutamicpyruvic transaminase (GPT), and alpha-fetoprotein (AFP). Total DNA of PBLs was assayed for ΔmtDNA4.9kb, mtDNA copy numbers and MTHFR C677T polymorphism by use of real-time PCR. Results of the part Ⅰstudy revealed that 60% of HCC patients were in marginal folate deficiency (serum folate level < 14 nM), and 19 % of HCC patients were in hyperhomocysteinmia. Serum B12 level of HCC patients was significantly and positively correlated with AFP level (r = 0.2657, P = 0.0119). Serum folate concentration was inversely correlated with HCC tumor size (r = -0.2227, P = 0.0349) and tumor numbers ( r = -0.2742, P = 0.0089). By TNM classification of HCC progression, lower level serum folate and higher concentrations of B12 and GOT levels were associated with higher degree of HCC progression. After the adjustment of multiple factors, odds ratio of large- sized tumor (>5 cm) and multiple tumors were 4.422 (95% CI = 1.272- 15.373) and 6.375 (95% CI = 1.117 -36.391) in HCC patients with low serum folate level (< 14 nM) compared to those in adequate folate level. The odds ratio of tumor aggressiveness (T>2 and stage >Ⅱ) was 6.148 (95%C.I. = 1.105-34.202) and 6.375 (95%C.I. = 1.117-36.391) in HCC patients in low serum folate level (< 14 nM) compared to those in adequate folate level MTHFR C677T polymorphism did not modify risks of tumor progression in HCC patients.Result of part Ⅱ study showed that ΔmtDNA4.9kb of HCC group was siginificantly higher than those of the controls. mtDNA large deletion significantly correlated with serum folate (r = -0.3148, P = 0.0001) and lymphocytic folate (r = -0.1559, P = 0.0411) , serum B12 (r = 0.2287, P = 0.0023) , GOT (r = 0.2059, P = 0.0063) and AFP levels (r = 0.2118, P = 0.0049). The odds ratio of HCC in the group with high ΔmtDNA4.9kb (ΔCt ≧5.32) was 5 compared to those with low ΔmtDNA4.9kb (ΔCt ≧2.535). For those with high mtDNA mutation and low serum folate level , the OR of HCC was 13.62 (95% C.I. = 1.453-122.85) compared to the adequate folate group. Control subjects carrying 677TT genotype had lower level of serum folate and higher level of homocysteine compared with subjects carrying 677CC genotype. HCC patients with 677TT genotype had significantly lower levels of ΔmtDNA4.9kb as compared to those carrying with 677CC genotype. The odds ratio of HCC was 0.016 (95%C.I. = 0.01-0.344) and 0.013 (95%C.I. = 0.001-0.539) in subjects carrying with 677TT compared to those carrying on 677CC in low serum folate status (< 14 nM) or high ΔmtDNA4.9kb (ΔCt ≧5.32) frequency. In summary, we found that the prevalence of folate deficiency was 60% in HCC patient. Low serum folate (< 14 nM) could predict the cancer risk of HCC. Incresed accumulation of ΔmtDNA4.9kb in PBLs was found in HCC patients. The odds ratio of HCC by high ΔmtDNA4.9kb frequency and low serum folate level was 13 (OR = 13.62, 95% C.I. = 1.453-122.85). MTHFR 677TT genotype modulated levels of mtDNA mutation in PBLs of HCC patients. OR of HCC for those with 677TT genotype was significantly reduced, suggesting that 677TT genotype may have a protection against HCC progression. The mechanism is unclear and requires more studies.

中文摘要………………………………………………………………..Ⅰ
英文摘要………………………………………………………………..Ⅲ
致謝……………………………………………………………………..Ⅴ
表目錄…………………………………………………………………..Ⅹ
第一章 前言………………………………………………………........ 1
第二章 文獻回顧…………………………………………………........ 2
ㄧ、肝細胞癌 (hepatocellular carcinoma；HCC)…………….. … 2
二、葉酸營養狀態與肝臟疾病的相關性……………………….....… 3
三、葉酸營養不良與肝臟腫瘤的相關機制………………………….. 4
四、血清維生素B12濃度與肝臟疾病之相關性…………………….. 5
五、維生素B12結合蛋白濃度與肝癌的相關性…………………….. 6
六、粒線體基因突變與人體肝細胞癌化的指標…………………..… 7
七、粒線體DNA的突變機制………………………………………… 8
八、粒線體DNA變異與肝炎,肝癌以及葉酸營養之相關性……….. 9
九、甲醯基四氫葉酸還原酶(methylenetetrahydrofolate reductase,
MTHFR) 基因多型性對單碳代謝的影響……………….....….11
十、MTHFR C677T基因多型性對癌症的影響………………..…..12
十一、MTHFR C677T基因多型性與肝癌的相關性…………….…13
十二、研究目的………………………………………………………13

第三章 實驗材料與方法………………………………….…………..14
一、實驗設計…………………………………………………………14
二、研究對象…………………………………………………………14
三、研究方法………………………………………………...……….15
1.血樣收集與單核淋巴球分離保存………………………………..15
2.淋巴球DNA之萃取…………………………………….………...16
3.淋巴球粒線體DNA 4977 bp大片段斷損檢測……….…………16
4.粒線體DNA拷貝數測定……………………………………….…17
5.引子與TaqMan探針的選擇………………………………………17
6.MTHFR C677T基因多型性分析……………………………..…18
7.淋巴球葉酸濃度分析…………………………………………..…19
(1)葉酸的萃取………………………………………………….…19
(2)葉酸濃度測定…………………………..…………………..…19
四、 統計分析……………………………………………………..…20

第四章 結果……………………………………………….…………..21Ⅰ: 單碳代謝生化分子 (葉酸、B12及同半胱胺酸)、MTHFR C677T
基因多型性與肝癌腫瘤進化之相關性……………………..……21一、肝癌組受試者基本資料分析……………………………………21
二、血清葉酸濃度與肝功能及肝細胞癌腫瘤進化程度的相關性…22
三、血清同半胱胺酸濃度對肝損傷指標與肝癌腫瘤進化的影響…24
四、血清維生素B12濃度對肝損傷指標與肝癌腫瘤進化的影響….26
五、單碳代謝相關生化分子 (葉酸、B12及同半胱胺酸)、肝臟損傷
指標與肝腫瘤進化指標之相關性………………………………28
六、單碳代謝相關生化分子 (葉酸、B12及同半胱胺酸)與肝臟損傷指標GOT與GPT以及肝腫瘤指標AFP之相關性……….…28
七、單碳代謝相關生化分子 (葉酸、B12及同半胱胺酸)與肝腫瘤體積與腫瘤顆粒數之相關性………………………………………29
八、腫瘤進化參數與單碳代謝生化分子 (葉酸、B12及同半胱胺酸)、肝損傷指標與MTHFR C677T基因多型性之相關……………30
九、以MTHFR C677T 基因型預測肝腫瘤大小增大、腫瘤顆粒數上升與癌轉移發生危險率…………………………………………31
十、血清葉酸濃度對肝癌進化之相對危險率影響…………………31

Ⅱ: 淋巴球粒線體DNA變異、單碳代謝生化分子 (葉酸、B12及同
半胱胺酸)與肝癌罹患率的相關性……………………………....32
一、受試者基本資料分析………………………………..………...32
二、受試者之淋巴球粒線體DNA變異性觀察…………..……….33
三、淋巴球粒線體DNA斷損量 (ΔmtDNA4.9kb) 與單碳代謝生化分子(血清與淋巴球葉酸、B12及同半胱胺酸)、MTHFR C677T基因多型性及肝功能損傷指標的相關性……………………..33
四、淋巴球粒線體DNA拷貝數與單碳代謝生化分子 (血清與淋巴球葉酸、B12及同半胱胺酸)、MTHFR C677T 基因多型性與肝功能損傷指標的相關性……………………………………..…35
五、淋巴球粒線體DNA變異、單碳代謝生化分子 (葉酸、B12及同半胱胺酸)、MTHFR C677T 基因多型性與肝功能損傷指標之相關性…………………………………………………………..36
六、淋巴球粒線體DNA大片段斷損、單碳代謝生化分子 (葉酸、B12及同半胱胺酸)、MTHFR C677T 基因多型性與肝功能損傷指標之相關性……………………………..……………………36
七、使用複邏輯回歸模式以淋巴球粒線體DNA大片段斷損預測肝癌罹患危險率……………………………………..……………37
八、使用複邏輯回歸模式以淋巴球葉酸濃度預測肝癌罹患危險率…………………………………………………………..……37
九、使用複邏輯回歸模式以血清葉酸與維生素B12濃度預測肝癌罹 患危險率……………………………………………………..…38
十、MTHFR C677T基因型分組間單碳代謝分子濃度、淋巴球粒線體DNA變異與肝功能損傷指標相關性…...……………….…38
十一、淋巴球粒線體DNA大片段斷損與血清葉酸濃度交互作用對肝癌罹患率的影響…………………………………………..39
十二、淋巴球葉酸濃度、血清維生素B12濃度與MTHFR C677T基因型對肝癌罹患率的影響………………………………..…40

第五章 討論……………………………………………………………42
一、肝癌患者葉酸缺乏營養狀態普遍性……………………………42
二、低血清葉酸濃度與肝損傷指標GOT、GPT的相關性……..….42
三、低葉酸營養狀態對肝腫瘤體大小增加、多腫瘤顆粒數與肝腫瘤
進化的影響…………………………………………………..…42
四、高血清維生素B12濃度對多腫瘤顆粒數與肝腫瘤進化的影響45
五、MTHFR C677T基因多型性對降低肝腫瘤增生具有保護性..45
六、淋巴球粒線體DNA變異性與單碳代謝分子濃度相關性…..…46
七、淋巴球粒線體DNA大片段斷損可反映肝功能損傷指數...…46
八、高淋巴球粒線體DNA大片段斷損顯著增加肝癌罹患危險率47
九、受試者MTHFR C677T 基因多型性發生頻率…………....…48
十、MTHFR C677T基因多型性與肝癌罹患相關性…………..…49

第六章 結論……………………………………………………………50
參考文獻………………………………………………………………..81








表目錄
表一 、肝癌組受試者基本資料分析……………………………........51
表二、血清葉酸濃度與營養生化指標及單碳代謝分子的相關性…..53
表三、血清葉酸濃度與肝功能及肝細胞癌腫瘤進化程度的相關性..54
表四、血清同半胱胺酸濃度對營養生化指標及單碳代謝分子的相關55
表五、血清同半胱胺酸濃度對肝損傷指標與肝癌腫瘤進化的影響…56
表六、血清維生素B12濃度營養生化指標及單碳代謝分子的相關性57
表七、血清維生素B12濃度對肝損傷指標與肝癌腫瘤進化的影響…58
表八、單碳代謝生化分子 (葉酸、B12及同半胱胺酸)與肝臟損傷指標之相關性………………………………………………………..59
表九、單碳代謝生化分子 (葉酸、B12及同半胱胺酸)與肝腫瘤進化指標之相關性……………………………………………………..60
表十、單碳代謝生化分子 (葉酸、B12及同半胱胺酸)與肝臟損傷指
GOT與GPT以及肝腫瘤指標AFP之相關性……………….61
表十一、單碳代謝生化分子 (葉酸、B12及同半胱胺酸)與肝腫瘤體積與腫瘤顆粒數之相關性…………………………………..…62
表十二、腫瘤進化參數與肝損傷指標之相關性………………..….…63
表十三、腫瘤進化參數與單碳代謝生化分子 (葉酸、B12及同半胱胺酸)、MTHFR C677T基因多型性之相關性…………….…64
表十四、MTHFR C677T基因多型性對肝腫瘤惡化之相對危險率影響....................................................................................65
表十五、血清葉酸濃度對肝癌進化之相對危險率影響……………..66
表十六、180位受試者基本資料分析…………………………………67
表十七、淋巴球粒線體DNA大片段斷損對營養生化指標及肝臟病變的相關性……………………………………………………..68
表十八、淋巴球粒線體DNA大片段斷損對肝功能指標及單碳代謝分子的相關性…………………………………………..………69
表十九、淋巴球粒線體DNA拷貝數對營養生化指標及肝臟病變的相關性………………………………………………..…………70
表二十、淋巴球粒線體DNA拷貝數對肝功能指標及單碳代謝分子的
相關性……………………………………………………..…71
表二十一、淋巴球粒線體DNA大片段斷損與肝功能指標相關性….72
表二十二、淋巴球粒線體DNA大片段斷損與單碳代謝分子濃度相關
性………………………………………………………..…73
表二十三、總體受試者、對照組與病例組內淋巴球粒線體DNA斷損
量與單碳代謝分子濃度、肝功能損傷指標與肝癌指標之相
關性……………………………………………………..…74
表二十四、使用複邏輯式回歸以淋巴球粒線體DNA變異預測HCC罹患率…………………………………………………..…75
表二十五、使用複邏輯式回歸以淋巴球葉酸濃度預測HCC罹患率…………………………………………………………..76
表二十六、使用複邏輯式回歸以血清葉酸濃度與維生素B12濃度預測
HCC罹患率………………………………………………77
表二十七、MTHFR C677T基因型分組間單碳代謝分子濃度、淋巴
球粒線體DNA變異與肝功能損傷指標相關性………...78
表二十八、淋巴球粒線體DNA大片段斷損與血清葉酸濃度交互作用
對肝癌罹患率的影響…………………………………..…79
表二十九、淋巴球葉酸濃度、血清維生素B12濃度與MTHFR C677T基因型對肝癌罹患率的影響…………………………..…80

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