臺灣博碩士論文加值系統

摘要
對於已接受治療的癌症病患而言，如何有效地在早期診斷出病患是否有癌細胞的轉移，一直是生物醫學研究中所重視的課題。而對偶基因異結合型的缺失（loss of heterozygosity, LOH）與微衛星不穩定性（microsatellite instability, MSI）均代表癌細胞內累積了許多遺傳變異而造成基因組不穩定的現象。本研究的目的是要在癌症病患的血漿DNA中，尋找和腫瘤組織DNA中LOH或MSI變異形式相符的微衛星標記（microsatellite markers）作為偵測癌細胞微轉移的生物標記，在本實驗中共使用18組微衛星標記分別針對肝癌、胃癌和乳癌病患取得其腫瘤組織、正常組織、血球或血漿DNA，利用螢光PCR-毛細管電泳的方法進行微衛星DNA的分析。而依據各個微衛星標記的異結合型比例、在腫瘤組織中發生LOH的比例及在血漿DNA中偵測到LOH的比例，分別篩選出在肝癌、胃癌和乳癌中的三種等級之微衛星標記。比較三種等級的標記在癌症病患腫瘤組織及血漿DNA中可偵測到LOH現象的比例，結果在乳癌中已找到第一等級的微衛星標記LPL、D16S413和TP53適合作為偵測乳癌癌細胞微轉移的生物標記，而在肝癌及胃癌中則需要再尋找更相關的微衛星標記。此外，比較乳癌病患的病理資料顯示在血漿DNA中能否偵測到LOH現象與腫瘤相關的病理資料均無顯著差異。而利用血漿進行LOH分析的偵測方法與臨床的淋巴腺病理切片方法相比較，發現利用血漿DNA進行微衛星分析的方法具有較高的偵測能力。所以，從本實驗結果中，提供了一個較無侵襲性又兼具專一性的檢驗方法，可應用於早期偵測癌細胞及作為癌症病患預後追蹤的參考依據。

Abstract
Early diagnosis of micrometastasis of cancer patients is always the major topic in cancer clinic. It is reasonable to assume that if we can detect cancer cells in peripheral blood of cancer patients, implying that they have high probability to have cancer metastasis. The phenomena of loss of heterozygosity (LOH) and microsatellite instability (MSI) in cancer cells are derived from the accumulation of genetic alterations. Both have been widely accepted as biomarkers of cancer cells. For the purpose of identifying the biomarkers for detecting micrometastasis of cancer cells, we designed the present study to determine the presence of tumor DNA in the plasma of patients with hepatocellular carcinomas (HCC), gastric cancer (GC), and breast cancer (BC), characterized by LOH in 18 microsatellite markers. DNA extracted from tumor tissues, neighboring normal tissues, blood cells and plasma of cancer patients was amplified with fluorescent primers specific for the microsatellite markers. PCR amplicons were separated on the ABI-3100 16 capillary array electrophoresis instrument and analyzed with GeneScan analysis software v.3.7. According to the ratios of heterozygosity, LOH in tumor and plasma DNA, microsatellite markers were classified as four grades. Markers with heterozygosity≧80 %, LOH in tumor DNA≧50 %, and LOH in plasma DNA≧30 % were classified as the first grade; those with heterozygosity≧80 %, LOH in tumor DNA≧50 %, and LOH in plasma DNA≦30 % were classified as the second grade; those with heterozygosity≧80 % and LOH in tumor DNA≦50 % were classified as the third grade; while those with heterozygosity≦80 % were classified as the fourth grade, which were considered as unsuitable markers. Three markers (LPL, D16S413 and TP53) were classified as the first grade markers in BC, which together detected 85.3 % (29/34) of tumor DNA with LOH, and 41.2 % (14/34) of plasma DNA with LOH. In HCC, TP53 were the only first grade marker, which detected 44.9 % (22/49) of tumor DNA with LOH, and 24.5 % (12/49) of plasma DNA. None was qualified as the first and second grade markers in GC. No association was found between the presence of LOH in plasma and clinicopathological parameters, such as tumor size, tumor stage, tumor grade, the expression of estrogen receptor, progesterone receptor, c-erbB-2 oncoprotein and p53. However, our method was significantly better than dissected lymph node histochemical method in detecting micrometastasis. As a conclusion, we have provided a specific and noninvasive method, which can be applied as a diagnostic technique for early diagnosis and prognostic follow-up tests for cancer.

目次
英文摘要………………………………………………………………VII
中文摘要………………………………………………………………IX
導論…………………………………………………………………… 1
材料與方法……………………………………………………………18
結果……………………………………………………………………23
討論……………………………………………………………………34
參考文獻………………………………………………………………48
圖………………………………………………………………………57
表………………………………………………………………………70

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