臺灣博碩士論文加值系統

結腸直腸癌(CRC)於2015年在世界為第三常見的癌症，同時在癌症死亡率也排名第三。大約有百分之六十的結腸直腸癌病人可以透過手術、化學藥物、放射線及標靶藥物治療，然而病患常因為復發而無法痊癒。先前的研究指出，癌症幹細胞主要有自我更新及化療藥物抗性，傳統的癌症治療通常只消滅已分化的癌細胞而非癌症幹細胞，因此常引起復發。癌症幹細胞標靶治療不僅可消除癌症幹細胞，體內剩餘的癌細胞亦可受到藥物影響而死亡，因此，傳統治療方式與癌症幹細胞標靶治療的結合應用或許可有效治療癌症。實驗室先前利用微陣列分析方式篩選出了胺基胜肽酶A，然而，此分子對於結腸直腸癌的轉移機制仍未清楚。透過酵素活性抑制試驗，實驗室由LOPAC 1280平台篩選出了一種藥物compound A，可有效抑制胺基胜肽酶A的活性，由實驗室先前的研究發現，compound A可以抑制結腸直腸癌細胞的移動、侵襲及聚球能力，因此，我們將探討compound A是透過哪一機制去抑制結腸直腸癌幹細胞的生成。透過MTS assay及Clonogenic assay得知，compound A可抑制細胞短期與長期的增生，並由流式細胞儀的分析得知compound A可使細胞週期停滯於G0/G1期，更進一步探討，compound A可抑制cyclin D1、p21、p27的表現，對於細胞增生的調控，推測compound A可能透過抑制ERK及β-catenin達到調控細胞週期的效果。利用sphere formation assay，初步得知compound A可明顯抑制癌症幹細胞基因Nanog及ENPEP基因的表現量。同時，在動物實驗中觀察到了餵予小鼠compound A，腫瘤的大小相較餵予水的組別小。綜上所述，目前實驗的結果可知compound A在細胞及動物實驗都有抑制癌症細胞生長的效果，但是對癌症幹細胞抑制機轉仍有待進一步的探討。

Colorectal cancer (CRC) is the third most common cancer in the world and ranked third in cancer relating deaths in 2015. Approximately sixty percent of CRC patients can be treated by surgery, chemotherapy, radiation therapy and targeted therapy. However, CRC is seldom cured due to recurrence. Previous studies indicates that cancer stem cells (CSCs) are responsible for self-renewal and resistance to cytotoxic agents. Traditional cancer therapies usually kill differentiated cancer cells but not CSCs, and often lead to relapse. Meanwhile, CSC-targeted therapies can eliminate CSCs, and the remaining cancer cells will gradually die out. Therefore, combination of traditional and CSC-targeted therapies should clear CSCs and differentiated cancer cells. Aminopeptidase A (ENPEP) was selected from microarray analysis because its role in CRC metastasis is still unknown. By enzymatic activity inhibition assay, we screened LOPAC 1280 system and selected compound A as a potent inhibitor of aminopeptidase A activity. Compound A was shown to inhibit cell migration, invasion and sphere formation ability in CRC cells. We tried to determine the inhibitory mechanism of CRC stem cells by compound A. In MTS assay and clonogenic assay, we found that compound A can inhibit short term and long term proliferation. Next, our data showed that compound A can arrest cell cycle at G0/G1 by flow cytometry. Furthermore, cyclin D1, p21 and p27 were downregulated with compound A treatment shown by Western blot. We also demonstrated that compound A may inhibit ERK and beta-catenin expression to induce cell cycle arrest. Through sphere formation assay, compound A can inhibit cancer stem cell genes like Nanog and ENPEP expression. Finally, we observed that mice fed with compound A had smaller tumor size compared with control group. In conclusion, our data evidenced that compound A inhibits cell proliferation both in vitro and in vivo. Yet the cancer stem cell inhibitory mechanism of compound A needs to be further studied.

致謝..............................i
中文摘要.........................iii
英文摘要..........................iv
目錄..............................vi
第一章、緒論......................................... ..1
第一節、結腸直腸癌 (Colorectal cancer).................. 1
第二節、細胞週期 (Cell cycle)........................... 3
第三節、癌症幹細胞 (Cancer stem cell)................... 6
第四節、Aminopeptidase A (ENPEP)簡介................... 7
第五節、Compound A簡介................................. 8
第六節、研究動機與目的...................................9
第二章、材料與方法......................................10
第一節、實驗材料........................................10
第二節、實驗方法........................................28
第三章、實驗結果........................................43
1.構築之ENPEP 1809△大量表現之質體轉染於293T細胞中並無APA蛋白酵素活性...............................................43
2.Compound A 可以抑制SW480及HT29結腸直腸癌細胞增生.......44
3.Compound A可使SW480細胞週期停滯(arrest)，停留於G0/G1期.45
4.Compound A抑制APA活性並非依賴Calcium之抑制............45
5.Compound A可以抑制SW480細胞株之β-catenin、ERK訊號傳遞路徑...................................................46
6.Compound A可抑制癌症幹細胞相關基因表現................47
7.Compound A在斑馬魚實驗平台可抑制細胞增生..............48
8.不同劑量的compound A可抑制小鼠結腸直腸癌細胞株CT26在BALB/c小鼠中的腫瘤生長......................................48
9.0.07mg/kg劑量之compound A可抑制小鼠結腸直腸癌細胞株於BALB/c小鼠中的腫瘤生長......................................49
第四章、實驗討論......................................50
第五章、參考文獻......................................57
第六章、實驗結果圖表...................................62
第七章、附錄..........................................81

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