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博碩士論文 etd-0727101-155454 詳細資訊
Title page for etd-0727101-155454
論文名稱
Title
隨機放大多型性DNA對非小細胞肺癌 基因不穩定之探討
Genomic instability in NSCLC detected by RAPD
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
61
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2001-07-25
繳交日期
Date of Submission
2001-07-27
關鍵字
Keywords
肺癌、基因不穩定
NSCLC, Genomic instability, RAPD
統計
Statistics
本論文已被瀏覽 5641 次,被下載 11297
The thesis/dissertation has been browsed 5641 times, has been downloaded 11297 times.
中文摘要
摘要
肺癌目前是台灣十大癌症死亡因素之一,每年罹患肺癌而死亡的人數達五千人之多。高居不下的死亡率和預後不佳的情形,更突顯肺癌的嚴重性。本研究利用DNA多型性隨機放大( Random Amplified Polymorphic DNA, RAPD ) 技術來分析肺癌細胞內基因不穩定現象
,進而探討肺癌形成過程中可能的基因變異情形,並找出與非小細胞肺癌發展有關的基因。在RAPD分析的正常與腫瘤檢體中,有三個或三個以上在這七個隨機的primers能被偵測到的人則定義為基因不穩定的人。而這七個隨機的primers中有四個曾是肺癌基因不穩定研究中被用來偵測的,另外三個則是我們新設計的primers。而RAPD分析的結果中,偵測到81.48% (22/27)非小細胞肺癌患者有基因不穩定情形。
本研究中以primer 6及primer 7的再現性最佳,且由primer 6及primer 7偵測到的非小細胞肺癌變異片段經轉殖定序方式,在染色體區找到可能的突變基因。而結果顯示在第二、四、六、十四及二十二對染色體區位DNA有擴增或缺失的情形。之後,經生物資訊搜尋比對人類基因庫後,發現一些致癌基因 (如RABL2B、c-fos、n-myc 和mas1)和抑癌基因 (AR, amphiregulin)且這些基因位於這些subclons的附近位置。
X2 test結果顯示基因不穩定與腫瘤分期 (stage)之間呈現統計意義上的相關性 ( p < 0.05),與其他的病理因素如病患年齡、腫瘤大小
、腫瘤轉移的有無、腫瘤分化程度、病人存活天數及組織型態種類等都不具統計上的意義( p > 0.05)。



Abstract
Abstract
Lung cancer is one of the most common cancer death in Taiwan. The dead population of lung cancer are over five thousands per year. High mortality and bad prognosis displayed the severity of the lung cancer. RAPD (Random Amplified Polymorphic DNA), a simple technique for the detection of genomic instability, has been used in this research. We inquire into genetic variation in carcinogenesis, and find out genes association with NSCLC. Three or more than three DNA fragment patterns of normal and lung cancer samples exhibited by RAPD from the seven arbitrary were classified as genomic instability. Four out of seven arbitrary primers have been used for lung cancer RAPD analysis and three of them were newly designed in this investigation. Analysis of genomic instability with these seven random primers in twenty-seven NSCLC patients revealed that 81.48% of NSCLC exhibited genomic instability.
The RAPD reproducibilities of primer 6 and primer 7 were the best among the seven primers used in this study. Therefore, the variable DNA fragments of primer 6 and primer 7 in RAPD analysis were subcloned and sequenced for the study of the possible mutated genes in NSCLC.
Results showed that DNA gains or losses were found in chromosomes 2, 4, 6, 14 and 22. After bioinformatic searching and alignments with human Genebank, some oncogenes (such as RABL2B, c-fos, n-myc and mas1)
and tumor suppressor gene (AR) were found located nearby the locus of
these subclones.
Genomic instability was investigated in relation with the clinical-pathological features such as age, stage, tumor size, metastasis, differential status, survival days and cancer types. Results, evaluated by the X2 test, were not significant except tumor stage.

目次 Table of Contents
目錄

中文摘要 ………………………………………………… i
英文摘要 …………………………………………………… ii
壹、 緒論 ………………………………………………… 1
貳、研究策略及材料方法
一、研究策略 …………………………………………………… 10
二、材料與方法
(一) RAPD分析
1.檢體來源 ……………………………………………………. 12
2.DNA萃取 ……………………………………………………. 12
3.Random Primer的設計 ……………………………………… 13
4.聚合&#37238;鏈反應 (PCR) ……………………………………….. 13 5.DNA電泳分析 ………………………………………………. 15
(二)突變基因選殖(Cloning of pGEM&Ograve;-T Easy vector)
1.Competent cells 配製 …………………………………………. 15
2.重組DNA …………………………………………………….. 16
3. DH5α形質轉換 …………………………………………….. 16
4.重組DNA的篩選 …………………………………………… 17
(三) DNA定序分析
1. pGEM&Ograve;-T Easy vector primer設計 …………………………… 18
2.Cycle sequencing 聚合&#37238;鏈反應及酒精純化 ………………. 18
3.ABI PRISMTM 377DNA Sequencer操作 ……………………… 19
(四)生物資訊分析及統計分析 ………………………………… 21
參、結果 …………………………………………………. 22
肆、討論 ………………………………………………….. 26
伍、參考文獻 ………………………………………………… 31
陸、圖表
圖一、 RAPD圖示 ……………………………………………… 39
圖二、 P1~P7 primers偵測正常與肺癌組織的DNA型態 …….. 40
圖三、 P1~P7 primers對於非小細胞肺癌基因不穩定的偵測率 41
圖四、 P6 primer放大偵測得之肺癌組織DNA片段擴增情形及
P7 primer放大偵測得之肺癌組織DNA片段缺失情形 42
圖五、 經EcoRI Restriction enzyme篩選inserted DNA fragments 43
圖六、 P6 primer放大的變異片段經DNA定序分析之後呈現
simlple repeat sequences ……………………………… 44
表一、 RAPD分析非小細胞肺癌基因不穩定的結果 ………… 45
表二、 基因不穩定與各臨床病理因子相關統計分析 ………. 46
表三、基因不穩定和支氣管肺泡癌、鱗狀上皮肺癌及肺腺癌統計
分析及各類發生率 ……………………………………… 47
表四、RAPD分析 (P6 primer)偵測擴增片段得之染色體區位
和此區位鄰近相關的致癌基因及其功能 ……………… 48
表五、RAPD分析 (P7 primer)偵測缺失片段得之染色體區位
和此區位鄰近相關的抑癌基因及其功能 ……………… 49
柒、附錄
附錄一、放大變異DNA片段轉殖至pGEM&reg;T Easy vector … 50
附錄二、非小細胞肺癌患者之臨床病理因子對照表 ……….. 51
附錄三之一至之七、P1 ~P7 primers偵測正常與肺癌組織的DNA
型態 ……………………………………….. 52
附錄四之一、27組正常及肺癌組織檢體的DNA濃度 ……… 59
附錄四之二、RAPD分析所使用的7個primers之序列與濃度 60
附錄五之一至之二、P6 primer放大肺癌組織(10.12.15.23號)之
擴增片段序列比對結果 ……………….. 61

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