臺灣博碩士論文加值系統

大腸直腸癌 (colorectal cancer) 是目前全球盛行率以及死亡率極高的惡性腫瘤之一。在大腸直腸癌的形成與惡化過程中，基因變異扮演重要的角色，尤其以上皮細胞生長因子受體 (epidermal growth factor receptor；EGFR) 訊息傳遞路徑的基因變異最為常見，這些基因包括EGFR、KRAS、BRAF、PIK3CA等。此外，這些基因變異也會影響標靶藥物的成效；因此發展一個快速及簡便的方法偵測這些變異對於臨床診斷非常重要。本研究利用肽鍊核酸 (peptide nucleic acid；PNA) 探針，發展偵測BRAF V600E、PIK3CA E545K/D549N突變方法，搭配先前實驗室開發的KRAS 突變檢測，組成突變檢測套組，作為臨床預估治療效果的指標。研究結果BRAF、PIK3CA已建立偵測的方法，可以偵測到0.1%的突變率，除了用PNA probe偵測臨床檢體外，也利用先前實驗室已開發之single-probe 方法偵測檢體，作為本研究發展方法的比較。使用PNA probe 與single-probe兩個方法偵測50個大腸直腸癌癌檢體的結果，偵測到的KRAS突變分別為19 (38.0%) 與17 (34.0%) 個；BRAF V600E突變分別為9 (18.0%) 與5 (10.0%) 個；PIK3CA E545K/D549N突變分別為7 (14.0%) 與5 (10.0%) 個，這些結果都經過定序確認。由上述結果可知PNA probe 的靈敏度比single-probe 高。因此我們希望PNA probe偵測突變方法能在臨床實驗室使用。

Colorectal cancer (CRC) is a malignant disease with high prevalence and high mortality worldwide. During CRC progression and metastasis, genes mutation plays an important role. Mutations in genes of epidermal growth factor receptor (EGFR) signaling pathway are common in CRC. These mutated genes include EGFR, KRAS, BRAF, and PIK3CA. Furthermore, these gene mutations also affect the efficiency of EGFR targeted therapy. Therefore it is important to establish a rapid and convenient detection methods for these mutations. This study used peptide nucleic acid (PNA) probes to develop detection methods for BRAF (V600E) and PIK3CA (E545K and D549N) gene mutations. These tests will be combined with the KRAS mutation test that our laboratory have established before and assembled as a mutation detection panel for prediction of therapeutic efficacy. The results showed that our tests using PNA probes can detect 0.1% mutant in the wild-type background. In addition to PNA probe method, we applied another detection method using single-probe, which our laboratory have been establish before, for comparison. in 50 colorectal cancer samples, the method using PNA probe and the method using single-probe detected 19 (38.0%) and 17 (34.0%) KRAS mutations, 9 (18.0%) and 5 (10.0%) BRAF V600E mutations，and 7 (14.0%) and 5 (10.0%) PIK3CA E545K/D549N mutations, respectively. All the mutations were confirmed by direct sequencing. This results indicated that the method using PNA probe have higher sensitivity than the method using single probe. We hope that the PNA probe panels for mutation detection can be used in clinical laboratories.

指導教授推薦書
口試委員審定書
國家圖書館授權書
長庚大學授權書
致謝……………………………………………………………………..v
英文名詞縮寫對照…………………………………………………….vi
中文摘要….…………………………………………………………...vii
英文摘要………………………………………………………….......viii
目錄…………..………………………………………………………...ix
圖表目錄………………………………………………………………xii
第一章、 文獻回顧…………………………………………………..1
1.1 大腸直腸癌 (Colorectal cancer) …….…………………….......1
1.2 EGFR訊息傳遞路徑……………………………………….…..6
1.3 臨床治療難題與偵測平台開發…………………………...….10
1.4 基因突變的檢測方法……………………………………..…..10
1.5 研究動機………………………………………………………14
第二章、 實驗目的…………………………………………..……..15
第三章、 實驗設計與目標…………………………………..……..16
3.1 實驗設計………………………………………………..……16
3.2 研究目標…………………………………………………..…18
第四章、 材料與方法…………………………………………...….20
4.1實驗材料………………………………………………………...20
4.2細胞株………………………………………………….……......20
4.3大腸直腸癌組織檢體………………………………………..….21
4.4DNA萃取………………………………………………..….......21
4.5製備質體標準品…….…………………………………..…....…22
4.6利用single-probe方法測定基因型…………………………..…23
4.7利用PNA probe方法以non-clamping PCR條件測定基因型…24
4.8利用PNA probe方法以clamping PCR 條件測定測定基因型..24
第五章、 實驗結果………………………………………………....26
5.1建立偵測BRAF及PIK3CA基因突變的高靈敏度檢測平台….26
5.1.1BRAF及PIK3CA的引子及探針設計………………..………26
5.1.2以Non-clamping PCR條件測試PNA probe對野生型及突變型之Tm…………………………………………………..….….26
5.1.3設定合適的clamping PCR條件………………...……………27
5.1.4PNA probe以clamping PCR條件偵測大腸直腸癌組織檢體KRAS、BRAF及PIK3CA的基因突變……..……………27
5.1.5構築BRAF、PIK3CA突變及野生型基因型片段的質體….....28
5.2比較PNA probes與single-probes方法檢測大腸直腸檢體結果…………………………………………………………………28
5.2.1重現single-probe方法之結果…………………..…………...28
5.2.2以single-probe方法偵測大腸直腸癌組織檢體KRAS、BRAF及PIK3CA的基因突變…………………..…………………..29
5.2.3比較PNA probe與single-probe方法偵測大腸直腸癌組織基因突變數量…………………………………………………..29
5.2.4比較PNA probe與single-probe方法偵測大腸直腸癌組織基因突變的比例………………………………………………..30
第六章、 討論………………………………………………..……..31
第七章、 參考資料…………………………………………..……..33

圖表…………………………………………………………………....38
表一、帶有KRAS、 BRAF或PIK3CA突變之細胞株……..………….38
表二、已構築之BRAF、PIK3CA突變及野生型質體列表……..….…39
表三、使用single-probe、PNA probe檢測50個大腸直腸癌檢體所發現之突變基因....................40

圖一、BRAF、PIK3CA 引子及探針相對位置....................................42
圖二、PNA probe在non-clamping PCR條件下BRAF不同基因型之融離曲線結果……………………….....43
圖三、PNA probe在non-clamping PCR條件下PIK3CA不同基因型之融離曲線結果……………………………………………....44
圖四、PNA probe在clamping PCR條件測試偵測KRAS基因突變的偵測極限.................45
圖五、PNA probe在clamping PCR條件測試偵測BRAF基因突變的偵測極限.....................46
圖六、PNA probe在clamping PCR條件測試偵測PIK3CA基因突變的偵測極限...........................47
圖七、PNA probe在clamping PCR條件偵測大腸直腸癌臨床檢體與突變基因關係圖.......................48
圖八、定序確認構築之BRAF、PIK3CA突變及野生型的質體..........49
圖九、PNA probe在non-clamping條件偵測genomic DNA及plasmid DNA.............50
圖十、以single-probe偵測細胞株之基因突變.................................51
圖十一、比較PNA probe與single-probe兩種方法偵測大腸直腸癌組織突變基因的數量................52
圖十二、計算PNA probe與single-probe兩種方法偵測大腸直腸癌組織突變基因的比例..................53

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