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研究生:殷本惠
研究生(外文):Pen-Hui Yin
論文名稱:肝癌雌激素代謝基因多型性與粒線體DNA數目變異之研究
論文名稱(外文):Study on Polymorphisms of Estrogen-Metabolizing Genes and Alteration of Mitocondrial DNA Copy Number in Hepatocellular Carcinoma
指導教授:戚謹文
指導教授(外文):Chin-Wen Chi
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:2004
畢業學年度:92
語文別:中文
論文頁數:160
中文關鍵詞:肝癌雌激素多型性粒線體DNA
外文關鍵詞:hepatocellular carcinomaEstrogenPolymorphismMitocondrial DNA
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肝癌 (Hepatocellular carcinoma, HCC) 位居臺灣癌症死亡率的第一位,而許多證據顯示在肝癌的癌化過程中性荷爾蒙可能扮演某種角色。本論文發現三種雌激素代謝相關酵素的基因多型性,具有CYP17基因表現可能較高的A2對偶基因,較易被引發CYP1A1基因表現的MspI對偶基因,和產物酵素表現出較低活性的COMT基因L對偶基因會增加臺灣婦女罹患肝癌之危險性,而這三種多型性,與台灣地區男性罹患肝癌的危險性,並無統計上顯著的關聯。具有易被引發表現的MspI對偶基因的異合子個體其罹患肝癌之危險率(以及95% 信賴區間)為6.61(1.35, 32.43), 而具有MspI對偶基因同合子之個體其罹患肝癌之危險率為12.00(1.73, 83.46)。此外具有愈多危險基因型,罹患肝癌的機率愈高,具有兩種危險基因型之個體,其罹患肝癌的危險率為12.63(1.50, 106.37),而具有三種危險基因型之個體,其罹患肝癌的危險率為16.67(1.82, 152.77),這個結果顯示雌激素的代謝在女性肝癌形成過程中扮演重要的角色。本論文並發現肝癌組織中粒線體內DNA及呼吸鏈蛋白質的含量較其相對應非癌肝臟組織低,此外有22%的肝癌病患其粒線體DNA的 D-loop的位置發生了突變。進一步的探討調控粒線體生合成基因的表現發現,肝癌組織中peroxisome proliferators-activated receptor □ coactivator-1的表現量明顯的降低,但mitochondrial single-strand DNA binding protein的表現量卻是增加的。此外和沒有長期飲酒史的肝癌病人相比,有長期飲酒史的肝癌病人其非癌肝組織內粒線體DNA的數目明顯減少,相反地粒線體DNA中4977鹼基對刪除突變所佔的比例卻明顯增加。使用低濃度EtBr處理HA22T/VGH肝癌細胞株可降低細胞內粒線體DNA的數目,而粒線體DNA數目較低的細胞其ATP含量較少並且生長速率較緩慢,對肝臟內雌性激素的主要代謝物二甲氧雌二醇及紫杉醇、doxorubicin等抗癌藥物的感受性提高,粒線體DNA數目較低的HA22T/VGH肝癌細胞株經二甲氧雌二醇處理後含四套以上染色體的細胞所增加的比例較少,細胞內的活性氧分子也並不會增加,這些可能是因為粒線體DNA數目較低的細胞內抗氧化蛋白基因的表現量增加所導致的結果,而粒線體DNA數目較低的細胞內Bcl家族蛋白的表現量增加可能導致其對抗癌藥物的感受性提高,這些結果顯示雌激素代謝的差異會影響肝癌的形成過程,而粒線體生合成機制的缺失以及粒線體DNA的突變在肝癌形成過程中扮演重要角色,此外粒線體數目的變異會影響細胞對雌激素代謝物及其他藥物的感受性,因此雌激素代謝差異與粒線體DNA數目變異的交互作用可能影響肝癌的形成、臨床表現以及對抗癌藥物的感受性。
Hepatocellular carcinoma (HCC) is the leading cause of cancer death in Taiwan, and many studies suggest that sex hormone plays a critical role in the carcinogenesis of this cancer. In this thesis, it was found that the estrogen-metabolizing gene polymorphisms (CYP17 A2, CYP1A1 MspI, or COMT L) raised the risk of HCC in women but not in men. The risk of HCC was elevated in women harboring either heterozygous or homozygous variants of the CYP1A1 gene and the respective odds ratio (and 95% confidence interval) were 6.61 (1.35, 32.43) and 12.00 (1.73, 83.46). Moreover, it was found that the risk of HCC was increased in the female subjects harboring higher numbers of high-risk genotypes, but not in male subjects. The OR for female HCC associated with two putative high-risk genotypes was 12.63 (1.50, 106.37), and the OR for three putative high-risk genotypes was 16.67 (1.82, 152.77). These findings strongly suggest that estrogen metabolism plays a critical role in female hepatocarcinogenesis. In addition, it was observed that mitochondrial DNA (mtDNA) copy number and the content of mitochondrial respiratory proteins were reduced in HCCs as compared with corresponding non-tumorous livers. Moreover, 22% of HCCs carried a somatic mutation in the mtDNA D-loop region. Expression of the peroxisome proliferator-activated receptor □ coactivator-1 was significantly repressed in HCCs (P<0.005) while the expression of the mitochondrial single strand DNA binding protein was up-regulated indicating the regulation of mitochondria biogenesis is disturbed in HCC. The non-tumorous liver of the HCC patients with a long-term alcohol drinking history contained reduced mtDNA copy number (P<0.05) and higher level of the 4,977 bp-deleted mtDNA (P<0.05) as compared with non-alcohol patients. These observations suggest that reduced mtDNA copy number, impaired mitochondrial biogenesis and somatic mutations in mtDNA are important events during carcinogenesis of HCC. In addition, a cell model of hepatoma cells harboring reduced mtDNA copy number was established by treating a hepatoma cell line HA22T/VGH with low concentration of ethidium bromide. It was found that the hepatoma cells harboring reduced mtDNA copy number have lower ATP content, slower proliferation rate, and higher susceptibility to 2-methoxyestradiol (2-ME), taxol as well as doxorubicin, respectively. Moreover, it was found that the proportion of the polyploidy cells induced by 2-ME was decreased in the hepatoma cells harboring reduced mtDNA content, and the intracellular level of reactive oxygen species was not elevated by 2-ME treatment. These alterations may result from increased antioxidant gene expression in the hepatoma cells harboring reduced mtDNA level. On the other hand, increased expression of the Bcl-2 family proteins in the hepatoma cells harboring reduced mtDNA copy number may lead to an increase in the susceptivity to anticancer drugs. These findings suggest that the decrease in mtDNA copy number of hepatoma cells elevate the susceptibility to 2-ME and anticancer drugs. In summary, interaction between the alteration in estrogen metabolism and the decrease in mtDNA copy number affect the carcinogenesis, clinical manifestation, and drug susceptivity of HCC.
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