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系統識別號 U0026-0812200910411956
論文名稱(中文) 利用即時定量PCR與雷射顯微切割技術分析口 腔癌及癌前期組織中粒線體DNA 4977bp 斷裂之研究
論文名稱(英文) Studies of mitochondrial DNA 4977bp deletion in microdissected oral cancer and precancer lesions by Real-Time quantitative PCR
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 91
學期 2
出版年 92
研究生(中文) 周文彬
研究生(英文) Wen-Pin Chou
學號 t1690104
學位類別 碩士
語文別 中文
論文頁數 79頁
口試委員 指導教授-靳應臺
指導教授-謝達斌
口試委員-林立民
中文關鍵字 粒線體  口腔癌  即時定量聚合脢連鎖反應  雷射顯微切割 
英文關鍵字 RTQ-PCR  LMD  mitochondria  oral cancer 
學科別分類
中文摘要 在台灣,口腔癌死亡率已躍進成十大男性死亡癌症的第五位,且這些口腔癌
患者大部分皆有嚼食檳榔的習慣。近年來的研究指出,在許多癌症(包括口腔
癌)皆發現有mtDNA(粒線體DNA)變異存在,且這些mtDNA 變異的累積很
有可能是一個重要的致癌因子。我們利用雷射顯微切割儀(LMD)收集16 位有
嚼食檳榔口腔癌患者和9 位無嚼食檳榔口腔癌患者個人癌前期變化、腫瘤和其周
圍間質的組織,使用即時定量聚合脢連鎖反應(RTQ-PCR)分析這些不同變異組織
中4977bp 斷裂粒線體DNA(depleted mtDNA)的比例,平均4977bp depleted
mtDNA( 除以總mtDNA)的比例分別為腫瘤組織(0.112%)、癌前變化組織
(0.768%)、淋巴組織(0.001%)。我們發現所有患者(16/16)癌前變化和腫瘤
組織的4977bp depleted mtDNA 比例皆高於淋巴組織(分別是814 倍/162 倍);十
二個患者(12/16) 癌前變化組織中的4977bp depleted mtDNA 比例比腫瘤組織
高,而10/14 腫瘤和10/15 癌前變化組織比其間質細胞有較高之4977bp depleted
mtDNA 比例,在9 位無嚼食檳榔口腔癌患者中也得到相似的結果。此外無嚼食
檳榔口腔癌患者其平均年齡64±14.89 years 高於嚼食檳榔患者年齡為51±8.79
years,並且淋巴節組織之4977bp depleted mtDNA 比例也較高(NBQ: 0.00518%
±0.0013%;BQ:0.00141%±0.00054%,unpaired t test:p<0.05),暗示是否無嚼
食檳榔者可能因為先天遺傳上基因的缺陷而易罹患癌症。我們嘗試利用「clonally
selection」和「mtDNA dilution」的理論來解釋以上現象並藉由粒線體DNA 和核
DNA(mtDNA/nuDNA)相對定量的結果證實「mtDNA dilution」理論之可能性。
利用超長片段聚合脢連鎖反應的方法偵測口腔癌化組織是否存在不同長片段粒
線體DNA 斷裂,在一檢體中觀察到在癌前變化和腫瘤組織中有不同deletion type
存在。另外,經過定序的結果我們發現在患者檢體中存在一個粒線體DNA 264bp
斷裂的存在。將來本研究之結果會與臨床資料結合,希望能對臨床診斷上有所助益
英文摘要 Oral cancer is the fifth leading cause of cancer deaths among men in Taiwan and
is closely associated with areca quid chewing habit. Recent studies showed that
mitochondrial DNA (mtDNA) mutations occur in various tumors including oral
cancers and that the accumulation of mtDNA deletions could be an important
contributor during carcinogenesis. We have analyzed mtDNA deletions by pairwise
comparisons among oral cancer, precancerous cells and their adjacent submucosal
stroma tissues in 16 patients with areca quid history and 9 patients no areca quid
history using laser microdissection. Real-time quantitative PCR (RTQPCR) was
performed to detect and quantify mtDNA with the 4977 bp deletion in the
histologically defined specified cell groups. Relative quantitative analysis by
RTQPCR revealed that the average proportions of 4977 bp deleted mtDNA over total
mtDNA were 0.112%, 0.768%, and 0.001% in cancer, precancer cells, and
lymphocytes of lymph node biopsies respectively. All of the patients (16/16)
presented higher proportion of mtDNA with 4977 bp deletion in the lesions than that
in the lymphocytes, with average increases of 814 folds in cancer, and 162 folds in
precancer cells. A decrease in the proportion of deleted mtDNA was observed in 10
of 16 patients when the disease progressed from precancer to cancer lesions. 10 out
of 14 cancer tissues and 10 out of 15 precancer lesions exhibited higher the proportion
of 4977 bp-deleted mtDNA in the stroma cells than in the lesion cells, respectively.
The same results in 9 patients with no areca quid history were observed.
Interestingly, the ages and proportions of 4977 bp deleted mtDNA in lymph node of
patients with no areca quid history are higher then patients with areca quid history
(NBQ: 64±14.89 years, 0.00518%±0.0013%; BQ: 51±8.79 years , 0.00141%
±0.00054%, unpaired t test: p<0.05). This result may suggest patients with no
areca quid history get cancer more easily because of their genetic deficiency. We
tried to explain our results using clonally selection、mtDNA dilution hypothesis and
confirmed by mtDNA/nuDNA relative quantity. We also observed different length
deletions between paired precancer and cancer tissues in one frozen sample by
EL-PCR. By DNA sequencing, a short length 264bp deletion was detected in some
samples. All these results will compare with clinical data in future, hoping that these
have some advantages for clinical diagnosis.
論文目次 中文摘要-i
英文摘要-ii
誌謝-iii
目錄-iv
表目錄-vi
圖目錄-vii
符號及縮寫-viii
序論-1
材料與方法:
一、病人篩選和樣品收集-17
二、雷射顯微切割-18
三、DNA 粗萃取與純化-19
四、聚合脢連鎖反應(Polymerase chain reaction)-21
五、洋菜膠體電泳-22
六、引子設計-23
七、即時定量聚合脢連鎖反應-23
八、標準曲線的建立-25
九、粒線體DNA 4977bp 斷裂之相對定量-26
十、統計方法-27
十一、膠體中PCR 產物之回收及定序-28
十二、超長片段聚合脢連鎖反應(Extend-Long Polymerase chain reaction)-29
十三、核DNA 和粒線體DNA 之相對定量-32
結果
一、利用傳統聚合脢連鎖反應(Polymerase chain reaction)偵測口腔癌患者癌前變
化、腫瘤、淋巴節組織4977bp 斷裂之粒線體DNA-35
二、利用即時定量聚合脢連鎖反應(Real-Time quantitative PCR)建立標準曲線
及melting curve 之分析-35
三、利用即時定量聚合脢連鎖反應(Real-Time quantitative PCR)偵測16 位嚼
食檳榔口腔癌患者之4977bp 斷裂粒縣體DNA 相對定量結果-36
四、利用即時定量聚合脢連鎖反應(Real-Time quantitative PCR)偵測9 位無嚼
食檳榔口腔癌患者之4977bp 斷裂粒縣體DNA 相對定量結果-37
五、利用即時定量聚合脢連鎖反應(Real-Time quantitative PCR)偵測10 位嚼
食檳榔口腔癌患者之核DNA 與粒縣體DNA 相對定量結果-38
六、利用超長片段聚合脢連鎖反應(Extend-Long Polymerase chain reaction)偵
測口腔癌患者癌前變化與腫瘤組織之粒線體DNA 斷裂情形-39
七、膠體中PCR 產物之定序-40
討論-41
參考文獻-48
圖表-56
附錄-72
自述-79
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