臺灣博碩士論文加值系統

大腸直腸癌在台灣為最好發之惡性腫瘤之一,其發生率每年約有一萬人。許多研究指出藉由阻斷訊息傳遞路徑可抑制細胞異常增生。而標靶藥物爾必德舒(Cetuximab)為一表皮生長因子類似物,其証實能與表皮生長因子受體(EGFR)結合,進而抑制細胞增生使患者存活率增加。但爾必德舒使用在轉移性大腸直腸癌個案中若具有EGFR下游基因-Kirsten ras (KRAS)和v-raf murine sarcoma viral oncogene homolog B1 (BRAF) 的突變存在,其治療效果明顯不彰。2011年有研究指出微小核糖核酸-378 在 KRAS 突變型 與野生型基因中其表現量具有意義之差異性。而此微小核糖核-378 乃是具有調控 MAPK 途徑調控因子之一。據此,我們假設此微小核糖核酸-378 能調控 MAPK活化路徑,使將近50%KRAS或 BRAF突變之大腸直腸癌患者可提升標靶藥物治療效果。本篇以七株大腸直腸癌細胞株進行基因突變型態歸納做為實驗模型,觀察微小核糖核酸-378與標靶藥物反應敏感度之彼此相關性。實驗結果指出（一）KRAS或BRAF突變與否與微小核醣核酸-378表現量有高度相關性。（二）轉染微小核醣核酸-378後再添加標靶藥物,發現對所有突變型態細胞有效增加其對標靶藥物敏感性。（三）藉由微小核醣核酸-378 表現量 提升造成腫瘤細胞增生現象,間接証實 KRAS 與 BRAF 突變型大腸癌其實存在有不同 的潛在腫瘤發展機轉。研究結論,微小核糖核酸-378 的高表達對於提高標靶藥物作用性提供具體成果,未來可能成為在近五成的 KRAS 或 BRAF 突變之大腸直腸癌患者無 效治療上,重燃一新的治療契機。

Colorectal cancer (CRC) is the most common human malignancies in Taiwan, with incidences of 10 thousand people per year. A number of studies suggested that blocking abnormal cell proliferation by inhibiting the MAPK(mitogen-activated protein kinase) pathway could be a promising treatment method for CRC. Target drug “cetuximab”, an epidermal growth factor (EGF) competitor which inhibit cell proliferation has been shown to improve the survival rate. Earlier reports indicated that mutations in the genes such as KRAS and BRAF which are downstream of the EGFR lead to the deleterious activation MAPK pathway regardless of the EGFR antibody (Cetuximab) treatment. A previous report in 2011, down regulation of miR-378 was identified in CRC patients, who possesses KRAS mutations. miR-378 as one of the regulators of MAPK pathway. Herein, we aimed to compare expression levels of miR-378 in wild type and mutated CRC cell lines and further to study the role of miR-378 in Cetuximab responses. We used seven cell lines as experiment models. Our results indicated 1. The significant differences in expression of miR-378 when compare with KRAS or BRAF mutated cell and wild type cells. Further, 2. after transfected miR-378 to both the mutated and wild type cell lines, we found expression of miR-378 did increase cetuximab response rates as expected. 3. Base on the rate of cell proliferation that after transfected miR-378, the different mechanisms of tumor development could be revealed. Our findings might contribute to the development of a possible therapeutic strategy to block multiple oncogenes for near 50% of colorectal cancers contain with KRAS or BRAF mutations.

中文摘要 i
英文摘要 ii
誌謝 iii
目錄 iv
表目錄 vi
圖目錄 vii
第一章 緒論 1
1.1 大腸直腸癌緣起 1
1.2 有絲分裂原活化蛋白激酶(MAPK pathway) 4
1.3 標靶藥物-爾必得舒 5
1.4 微小核糖核酸 6
第二章 研究目標 9
第三章 實驗材料 10
3.1 細胞株 10
3.2 化學藥品與實驗材料 10
3.3 儀器設備 11
第四章 實驗方法 12
4.1 細胞培養 12
4.1.1 細胞培養條件 12
4.1.2 細胞冷凍保存及解凍 12
4.2 細胞株突變篩檢 13
4.2.1 抽取基因組DNA(Genomic DNA) 13
4.2.2 突變基因熱點區段聚合酶連鎖反應 13
4.2.3 基因定序 14
4.3 微小核糖核酸表現量偵測 14
4.3.1 抽取RNA 15
4.3.2 反轉錄 15
4.3.3 Taqman real-time PCR 16
4.3.4 轉染微小核糖核酸 17
4.4 細胞存活率分析 17
4.4.1 細胞凋亡分析 17
4.4.2 細胞存活率分析 18
4.5 統計方法 19


第五章 實驗結果 20
5.1 細胞株突變型態的分類 20
5.2 比較微小核糖核酸-378於原始細胞株的表現量 22
5.3 不同濃度爾必得舒對大腸直腸癌細胞株之毒性反應 23
5.4 比較大腸直腸癌細胞株轉染微小核糖核酸-378前後之生
長情形 25
5.5細胞轉染微小核糖核酸前後添加標靶藥物反應結果 26
第六章 實驗結論 29
參考文獻 33

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