臺灣博碩士論文加值系統

摘要
對偶基因不平衡所造成的LOH（loss of heterozygousity）是腫瘤發生過程中獨特的遺傳變異機制之一。Gene Scan偵測方式會受限於異結合比例，微衛星定量分析（QuMA）不僅能克服此問題，還能區分變異是放大或缺失。本實驗欲以QuMA建立偵測遺傳變異的系統，並與Gene Scan比較是否具一致性，進而取代之。沿用Gene Scan篩選的一級標記D17S849（17p）、D8S505（8q）和二級標記D16S3091（16q），對肝癌組織、血漿、血球進行相對定量，所用的內在校正有兩種：參考基因（β-actin）及螢光測量法(Fluorometry)所得DNA原始濃度。結果如下所述，（1）組織－β-actin為內在校正時，QuMA和Gene Scan結果相同的比例為：82.1％（D17S）、90％（D8S）、75％（D16S），兩種方法以 Kappa一致性分析，確實呈現高度符合的結果（p＝0.01、p＝1.6×10-4、p＝0.02）。Fluorometry為內在校正部分：60.7％（D17S）、53.3％（D8S）、66.7％（D16S）。（2）血漿－β-actin部分結果一樣的比例是33.3％（D17S）、65.2％（D8S）、46.7％（D16S）。Fluorometry部分35％（D17S）、27.3％（D8S）、66.7％（D16S）。本實驗發現β-actin當內在校正比較精確，Fluorometry不適合可能是未經過PCR反應過程。血漿結果差異甚大，可能是Gene Scan測量血漿微衛星DNA不是很靈敏，因為有許多檢體皆偵測不到，而且血漿半衰期很短，導致符合比例不高。（3）血球－QuMA測得肝癌與正常人的相對量進行判別分析，得到組別正確分類的機率是91.9%，可見正常人與癌症可以相對量區分。進一步分析三組微衛星以QuMA和Gene Scan檢測後，整體存活率及無病存活率皆無顯著關係，原因是當初Gene Scan僅根據組織、血漿中LOH的比例認定這三組標記，並沒有將預後的關係包含在內。本實驗還發現， Fluorometry測得的血漿原始濃度和轉移有顯著關係（p =0.0048）。本實驗證明QuMA確實不受限異結合比例、可快速地進行篩檢、操作又比Gene Scan方便，希望在未來能運用於癌症的早期診斷與預後評估。

Abstract
Genetic alteration plays an important role in cancer progression. Loss of heterozygousity (LOH), caused by allelic imbalance, is one of the distinctive genetic alteration mechanisms in the process of cancer progression. The way Gene Scan detects LOH is limited to the ratio of heterozygosity, while QuMA not only solves the problem, but it also differentiates whether a genetic alteration gains or loses. The following experiment is designed, by means of QuMA, to establish a system which can detect the genetic alteration, and to compare if the results correspond to those detected by Gene scan, in an attempt to substitute for Gene Scan. Used by Gene scan, the first rank of the markers, D17S849 (17p), D8S505 (8q), and the second rank of the marker, D16S3091 (16q), are adopted to perform relative quantification of tissue, plasma, and blood in liver cancer in the experiment. To the internal controls, β-actin, and the original density of DNA gained by Fluorometry are used for revision. The results are as follows: (1) in the experiment on tissue using β-actin as an internal control, 82.1% of results showed by D17S, 90％ by D8S, and 75％ by D16S accord with those by Gene Scan, revealing high correspondences based on Kappa coefficient of agreement（p＝0.01、p＝1.6×10-4、p＝0.02）.When the original density of DNA gained by Fluorometry is adopted as an internal control, the corresponding results are 60.7％ by D17S, 53.3％ by D8S, and 66.7％ by D16S. (2) In the experiment on plasma using β-actin as an internal control, the consistent results are 33.3％ conducted by D17S, 65.2％ by D8S, and 46.7％ by D16S, while the equivalent results in using the original density of DNA gained by Fluorometry as an internal control are changed to 35％ by D17S, 27.3％ by D8S and 66.7％ by D16S. The experiment indicates that β-actin used by QuMA as an internal control shows more accuracy than Fluorometry. The reason why Fluorometry is inappropriate might be that Fluorometry did not undergo the process of PCR reaction. The results in plasma by QuMA differ greatly from those by Gene Scan. The cause might be that Gene Scan was not sensitive to detect microsatellite DNA of plasma, because many samples were not detected by it, and the period of half-life of plasma was short, leading to low ratio of consistency. (3) In the experiment on blood, according to QuMA, the relative amount of people with lung cancer and that of people without the cancer are verified by discriminate analysis. The ratio of accuracy in discriminating the two classes of people amounts to 91.9% based on discriminant analysis. Therefore, whether people have lung cancer or not can be differentiated by the relative amount. In further analysis of the three markers used by QuMA and Gene scan, there is no obvious relationship between the overall survival and disease free survival to the markers. The reason is that Gene scan adopted the three markers according to the ratio of LOH in tissue and plasma, exclusive of the assessment of prognosis. Also, the experiment found out that the original density of DNA gained by Fluorometry is manifestly related to metastasis（p=0.0048）. The experiment verifies that QuMA is in fact not restrained to the ratio of heterozygosity, and is fast to screen. In addition, QuMA is more convenient for manipulation than Gene Scan. It is hoped that QuMA will be applied to detect cancer in its early stage and for prognosis in the future.

中文摘要 I
英文摘要 II
第一章 文獻回顧
第一節 癌症的發生 1
第二節 體液DNA 3
一、體液DNA的構造與來源 4
二、血漿/血清DNA與癌症 5
三、血漿/血清中微衛星DNA的變異 6
四、分析血漿/血清中微衛星DNA的限制　 7
五、體液DNA於醫學上的應用 8
第三節 癌症的分子診斷 8
一、突變基因及單一核酸多變性的檢測 9
二、檢測微衛星序列不穩定(MSI) 10
三、生物晶片 13
四、血清腫瘤標誌的檢測 15
五、蛋白質體與癌症的分子診斷 16
六、基因表現量的檢測 17
第四節 及時定量PCR（Real-Time PCR） 18
一、Real-Time PCR的發現與影響 19
二、Real-Time PCR與其它方法的比較 19
三、Real-Time PCR的高靈敏度 20
四、Real-Time PCR的應用 20
（一）偵測癌症預後的分子診斷 22
（二）快速評估癌症病患的治療選擇 23
（三）偵測血漿DNA 24
（四）突變分析 25
第五節 實驗目的 26
第二章 材料方法　　　 28
第三章 結果　　　　 38
第四章 討論　　　　 45
第五章 結論　　　　 51
第六章 參考文獻　　　 52
第七章 附表與圖　　 59

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