臺灣博碩士論文加值系統

K-ras 基因突變影響癌症病程與治療效果，大腸直腸癌中以抗表皮生長因子受體標靶治療藥物治療，但 K-ras 基因的突變導致治療效果不佳，因此檢測病患是否具有 K-ras 基因的突變可作為療程選擇及效果的指標。為了分析腫瘤，傳統上採取侵入性較高的手術來取得細胞或組織，但此方法對患者的身體帶來較大的負擔，採樣的次數亦受到限制，相對地抽血是侵入性相對較低的方式，血液中的循環腫瘤細胞 (circulating tumor cells, CTCs) 可作為臨床病人即時檢體，了解癌症病患病程進展以及治療藥物的選擇。本研究建立了利用 PowerMag 系統純化血液中的循環腫瘤細胞不經過核酸萃取直接使用細胞進行肽鏈核酸探針 (peptide nucleic acid, PNA) 即時聚合酶連鎖反應 (PNA-PCR) 檢測 K-ras 基因突變。不經過核酸萃取直接使用細胞的情況下 PNA-PCR 可偵測到 10 顆具有 G34A、G34T、G34C、G35A、G35T、G35C、G37T 與 G38A K-ras 基因突變的細胞，上述八種突變型涵蓋了 codon 12 與 13 中約 90 % 的突變。將具有 K-ras 基因突變的細胞加入 5000 顆白血球或健康人血液經 PowerMag 系統處理後的產物中，模擬實際檢體情況，在有 5000 顆白血球或健康人血液經 PowerMag 系統處理後的產物中若有 10 顆具有 K-ras 基因突變的細胞亦可被偵測到。最後使用臨床大腸直腸癌病人的血液檢體進行初步測試，16 個檢體中有 2 個 PowerMag-PNA-PCR 檢測結果顯示其循環腫瘤細胞具有 K-ras 基因突變。以目前測試結果來說 PowerMag-PNA-PCR 可以提供侵入性較低的方法進行 K-ras 基因突變檢測，以利了解臨床病人的腫瘤情況與協助訂定治療方法。

K-ras mutation in the cancer cells is associated with the resistance to the anti-epidermal growth factor receptor (EGFR) targeted therapy, an innovative strategy for treatment of patients with colorectal cancer. Tumor tissues are the primary resource for K-ras mutation detection. However, tumor tissues are not available for the patients who are not suitable for surgical operation or who are under recurrence long after surgical operation. Circulating tumor cells (CTCs) provide an alternative resource to unveil the mutation status. In this study, we established a method (PowerMag-PNA-PCR) for the combined use of the PowerMag CTC isolation system and the peptide nucleic acid (PNA)-PCR to enrich CTCs from the cancer patients followed by cell-direct and rare cell detection common K-ras codon 12 and 13 mutations without DNA purification. A method for cell lysis without interference of the assay was established to facilitate cell-direct PNA-PCR analysis. As less as 10 cells carrying the K-ras mutation of G34A, G34T, G34C, G35A, G35T, G35C, G37T, and G38A that covered more than 90% of the codon 12 and 13 mutations can be revealed. To mimic the condition of clinical specimens, K-ras mutant cells were spiked into various numbers of white blood cell (WBC) or the PowerMag eluted cell fraction from the healthy donors’ peripheral blood. The detection limit of cell-direct PCR was reached with 10 K-ras mutant cells in a background of 5000 WBCs or the eluted cell fraction. A pilot study using the peripheral blood from the patients (n = 16) with colorectal cancer showed that 2 of the patients carried the K-ras gene mutation. The PowerMag-PNA-PCR assay provides a novel and non-invasive approach for K-ras mutation detection in CTC and facilitates the decision making of anti-EGFR targeted therapy.

指導教授推薦書
口試委員審定書
致謝 iii
中文摘要 iv
英文摘要 v
縮寫表 vi
目錄 vii
圖目錄 ix
表目錄 x
第一章、 緒論 1
1.1 K-ras 基因與其蛋白質功能 1
1.2 K-ras 基因突變造成蛋白質功能變異 1
1.3 K-ras基因在癌症上的意義 2
1.4 K-ras 基因突變在大腸直腸癌中的影響 3
1.5 K-ras基因突變檢測技術 4
1.6 周邊血液循環腫瘤細胞在臨床上的意義及其相關研究 5
1.7 利用負向篩選的方式分離周邊血液循環腫瘤細胞 6
1.8 實驗目的 8
第二章、 材料與方法 10
2.1 材料 10
2.2 方法 11
2.2.1 細胞培養 11
2.2.2 細胞株核酸純化 11
2.2.3 直接使用細胞進行 PNA-PCR 之偵測極限 12
2.2.4 健康人與臨床大腸直腸癌病人血液樣本收集 12
2.2.5 模擬實際檢體情況進行 K-ras 基因突變檢測 12
2.2.6 臨床大腸直腸癌病人血液檢體進行 K-ras 基因突變檢測 13
第三章、 結果 15
3.1 PNA-PCR 可以偵測不同突變型的 K-ras 基因 15
3.2 比較直接將細胞加入 PCR 反應中與使用 QIAGEN 萃取 DNA 進行 K-ras 基因突變檢測的效率 15
3.3 直接使用細胞進行 PNA-PCR 的偵測極限 15
3.4 將具有 K-ras 突變的細胞加入健康人全血經 PowerMag 系統處理後的產物中以模擬臨床檢體 16
3.5 經 PowerMag 系統處理後殘留不表現 EpCAM 的有核細胞對 PNA-PCR 檢測 K-ras 突變的影響 16
3.6 臨床大腸直腸癌病患血液檢體測試結果 17
第四章、 討論 18
參考文獻 22
圖表附件 29

 
圖目錄
圖一、K-RAS 訊號傳遞路徑 29
圖二、PNA-PCR 可以偵測不同突變型的 K-ras 基因 30
圖三、比較直接使用細胞與使用QIAGEN萃取 DNA 進行進行 K-ras 基因突變檢測的效率 31
圖四、直接使用細胞進行 PNA-PCR 的偵測極限 32
圖五、將具有K-ras 突變的細胞加入健康人全血經PowerMag系統處理後的產物中以模擬臨床檢體進行 K-ras 突變檢測 33
圖六、野生型 K-ras 細胞對 PNA-PCR 檢測 K-ras 突變的影響 35
圖七、臨床大腸直腸癌病患血液檢體 K-ras 基因突變測試結果 36

 
表目錄
表一、K-ras 基因突變與細胞株 38
表二、經 PowerMag 系統處理後殘留不表現 EpCAM 的有核細胞 數目 39
表三、臨床檢體分析結果 40
 

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