癌症是台灣十大死因之首，許多癌症仍缺乏方便、快速的早期篩檢方式。本研究探討近年發展出具有高專一性的新興臨床檢測用材料適體（Aptamer）進行十大癌症中之大腸癌與卵巢癌的辨識。適體為單股核酸，經由指數增幅型系統性配子演化法（Systematic evolution of ligands by exponential enrichment, SELEX）可篩選出具有特殊立體結構能與標的物具專一鍵結能力之適體。本研究與動力機械工程學系李國賓老師實驗室合作，利用其所開發之自動化微流體適體篩選系統篩選出大腸癌適體（HCT-8-17、HCT-8-34）與卵巢癌適體（Tx-Ov-02rc）。利用大腸癌細胞HCT-8與卵巢癌細胞OVCAR3、OVCAR4與小鼠纖維母細胞Balb/c 3T3進行適體鍵結試驗，驗證適體鍵結標的物之能力與專一性。本研究並誘導免疫缺損小鼠生成腫瘤，將Cy5螢光標定適體以尾靜脈注射方式注入小鼠體內，觀察適體於動物體內代謝途徑與腫瘤鍵結能力。結果顯示Tx-Ov-02rc對於卵巢癌細胞OVCAR3有較高的專一性，也可觀察到其鍵結於活體腫瘤。其餘適體則較不具專一性。另外，李國賓老師實驗室也開發出胜肽自動化微流體篩選系統並利用此系統篩選出大腸癌胜肽（HOLC-1、HOLC-2）與卵巢癌胜肽（Tp-Ov-3-1）。我們同樣以鍵結試驗確認經自動化系統篩選出之胜肽對標的物的專一性。試驗結果發現HOLC-1對大腸癌細胞HCT-8有較高的專一性；HOLC-2與Tp-Ov-3-1胜肽對細胞之專一性較低。同時，我們將胜肽Tp-Ov-3-1共軛紅螢光蛋白mCherry，觀察攜帶大分子之胜肽是否會影響其鍵結細胞之能力。由胜肽鍵結試驗結果發現攜帶mCherry之胜肽並不影響其鍵結細胞能力，未來，可將mCherry更換為具毒殺細胞能力之蛋白生成抗癌胜肽。綜合上述，適體與胜肽具有成為新興癌症篩檢試劑與抗癌藥物之潛力。

Cancer is the number one cause of death in Taiwan. Most of cancers have no convenient, rapid screening method. In this study, we evaluated the specificity of novel clinical cancer screening material, aptamer, in detecting colon cancer and ovarian cancer, which are among top ten cancers. Aptamers are single-stranded nucleic acids that specifically bind to target molecules by their 3-dimentional structure and can be selected through systematic evolution of ligands by exponential enrichment (SELEX) procedure. This study is a collaboration with Prof. Lee, Gwo-Bin who developed an automatically microfluidic aptamer system for screening several aptamers to detect colon cancer (HCT-8-17 and HCT-8-34) and ovarian cancer (Tx-Ov-02rc). First, we verified the specific binding between aptamers and HCT-8 colon cancer, OVCAR3 ovarian cancer and Balb/c 3T3 fibroblasts by aptamer binding assay. We found that ovarian cancer aptamer Tx-Ov-02rc can bind to OVCAR3. Furthermore, we induced tumor on an immunodeficient mouse and injected Cy5-labelled aptamers into the mouse via tail vein. The result shows that Tx-Ov-02rc can specifically bind to OVCAR3 in vitro and in vivo. Prof. Lee’s lab also developed an automatically microfluidic peptide screening system to screen several peptides including colon cancer-targeting peptides (HOLC-1 and HOLC-2), an ovarian cancer-targeting peptide (Tp-Ov-3-1). We evaluated these peptides’ specificity by peptide binding assay and found that HOLC-1 can bind to HCT-8. Finally, we conjugated red fluorescent protein mCherry to the peptide, Tx-Op-3-1. In order to investigate whether macromolecule will influence the binding ability of peptides. The result showed that mCherry-conjugated peptides possessed specific binding ability to ovarian cancer cells. In the future, we can replace mCherry protein into a protein with cell-killing ability to generate a cancer-specific drug. To sum up, aptamer and peptide can be a potential cancer diagnostic reagent and anti-cancer drug.

目錄
中文摘要 I
Abstract II
誌謝 III
縮寫表 IV
目錄 VI
表目錄 VII
圖目錄 VII
附錄目錄 VIII
壹、 前言 1
貳、 材料與方法 5
2.1 細胞培養 5
2.2 欲試驗之核酸與胜肽 6
2.3 固定貼附細胞之適體/胜肽鍵結試驗 7
2.4 懸浮細胞適體/胜肽鍵結試驗 8
2.5 腫瘤小鼠建立方法 8
2.6 非侵入式3D活體影像系統（in vivo Imaging System, IVIS） 10
2.7 細菌繼代培養 11
2.8 勝任細胞（Competent cell）製備 12
2.9 建構Tp-Ov-3-1共軛mCherry重組蛋白表現質體 12
2.10 表現、純化Tp-Ov-3-1共軛mCherry重組蛋白 15
2.11 Tx-Ov-02rc適體連接QdotTM 525之細胞鍵結試驗 16
參、 結果 17
3.1 適體鍵結細胞試驗 17
3.2 適體標定小鼠腫瘤試驗 18
3.3 胜肽鍵結細胞試驗 20
3.4 mCherry共軛Tp-Ov-3-1胜肽鍵結細胞試驗 21
3.5 Tx-Ov-02rc適體連接QdotTM 525之細胞鍵結試驗 21
肆、 討論 22
伍、 參考文獻 25
附錄 50

表目錄
表一、 本研究使用之適體與胜肽序列表 31

圖目錄
圖一、 適體/胜肽固定貼附細胞鍵結試驗流程示意圖 32
圖二、 懸浮細胞鍵結試驗流程示意圖 33
圖三、 固定貼附細胞之HCT-8-17、HCT-8-34適體鍵結試驗 34
圖四、 懸浮細胞之HCT-8-17適體鍵結試驗 35
圖五、 固定貼附細胞之Tx-Ov-02rc適體鍵結試驗 36
圖六、 懸浮細胞之Tx-Ov-02rc適體鍵結試驗 37
圖七、 注射Balb/c 3T3與HCT-8多細胞類球體 38
圖八、 大腸癌腫瘤小鼠之非侵入性3D活體影像 40
圖九、 注射Balb/c 3T3與OVCAR3多細胞類球體 41
圖十、 卵巢癌腫瘤小鼠之非侵入性3D活體影像 43
圖十一、 固定貼附細胞之HOLC-1、HOLC-2胜肽鍵結試驗 44
圖十二、 固定貼附細胞之Tp-Ov-3-1胜肽鍵結試驗 45
圖十三、 懸浮細胞之Tp-Ov-3-1胜肽鍵結試驗 46
圖十四、 mCherry共軛Tp-Ov-3-1胜肽之固定貼附細胞鍵結試驗 47
圖十五、 Tx-Ov-02rc適體連接QdotTM 525之貼附活細胞鍵結試驗 48
圖十六、 Tx-Ov-02rc適體連接QdotTM 525之固定貼附細胞鍵結試驗 49

附錄目錄
附表一、 適體抗體比較表 50
附表二、 小鼠腫瘤生成率 51
附圖一、 李國賓老師實驗室研發之自動化微流體系統晶片 52

1 Chiang, C. J., Chen, Y. C., Chen, C. J., You, S. L. & Lai, M. S. Cancer trends in Taiwan. Jpn. J. Clin. Oncol. 40, 897-904 (2010).
2 Mandel, J. S., Bond, J. H., Church, T. R., Snover, D. C., Bradley, G. M., Schuman, L. M. & Ederer, F. Reducing mortality from colorectal cancer by screening for fecal occult blood. New Engl. J. Med. 328, 1365-1371 (1993).
3 Arbyn, M., Buntinx, F., Ranst, M. V., Paraskevaidis, E., Martin-Hirsch, P. & Dillner, J. Virologic versus cytologic triage of women with equivocal Pap Smears: a meta-analysis of the accuracy To detect high-grade intraepithelial neoplasia. JNCI-J. Natl. Cancer I. 96, 280-293 (2004).
4 Lauterbur, P. C. All science is interdisciplinary—from magnetic moments to molecules to men (Nobel Lecture). Angew. Chem. Int. Edit. 44, 1004-1011 (2005).
5 Gambhir, S. S. Molecular imaging of cancer with positron emission tomography. Nat. Rev. Cancer 2, 683-693 (2002).
6 Song, K. M., Lee, S. & Ban, C. Aptamers and their biological applications. Sensors 12, 612-631 (2012).
7 Hamaguchi, N., Ellington, A. & Stanton, M. Aptamer beacons for the direct detection of proteins. Anal. Biochem. 294, 126-131 (2001).
8 O'Malley, R. P., Mariano, T. M., Siekierka, J. & Mathews, M. B. A mechanism for the control of protein synthesis by adenovirus VA RNAI. Cell 44, 391-400 (1986).
9 Sullenger, B. A., Gallardo, H. F., Ungers, G. E. & Gilboa, E. Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication. Cell 63, 601-608 (1990).
10 Vermeer, A. W. P. & Norde, W. The thermal stability of immunoglobulin: unfolding and aggregation of a multi-domain protein. Biophys. J. 78, 394-404 (2000).
11 Stoltenburg, R., Reinemann, C. & Strehlitz, B. SELEX—a (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomol. Eng. 24, 381-403 (2007).
12 Lakhin, A. V., Tarantul, V. Z. & Gening, L. V. Aptamers: problems, solutions and prospects. Acta Naturae 5, 34-43 (2013).
13 Farokhzad, O. C., Cheng, J., Teply, B. A., Sherifi, I., Jon, S., Kantoff, P. W., Richie, J. P. & Langer, R. Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo. P. Natl. A. Sci. 103, 6315-6320 (2006).
14 Ng, E. W. M., Shima, D. T., Calias, P., Cunningham, E. T., Guyer, D. R. & Adamis, A. P. Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nat. Rev. Drug Discov. 5, 123-132 (2006).
15 Gragoudas, E. S., Adamis, A. P., Cunningham, E. T., Feinsod, M. & Guyer, D. R. Pegaptanib for neovascular age-related macular degeneration. New Engl. J. Med. 351, 2805-2816 (2004).
16 Tuerk, C. & Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505-510 (1990).
17 Ellington, A. D. & Szostak, J. W. in vitro selection of RNA molecules that bind specific ligands. Nature 346, 818-822 (1990).
18 Uzawa, T., Tada, S., Wang, W. & Ito, Y. Expansion of the aptamer library from a "natural soup" to an "unnatural soup". Chem. Commun. 49, 1786-1795 (2013).
19 Reverdatto, S., Burz, D. S. & Shekhtman, A. Peptide aptamers: development and applications. Curr. Top. Med. Chem. 15, 1082-1101 (2015).
20 Smith, G. P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315 (1985).
21 Colas, P., Cohen, B., Jessen, T., Grishina, I., McCoy, J. & Brent, R. Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2. Nature 380, 548-550 (1996).
22 Byrne, J. D., Betancourt, T. & Brannon-Peppas, L. Active targeting schemes for nanoparticle systems in cancer therapeutics. Adv. Drug Deliver. Rev. 60, 1615-1626 (2008).
23 Zhou, G., Wilson, G., Hebbard, L., Duan, W., Liddle, C., George, J. & Qiao, L. Aptamers: a promising chemical antibody for cancer therapy. Oncotarget 7, 13446-13463 (2016).
24 Liu, K. C., Lin, B. S. & Lan, X. P. Aptamers: a promising tool for cancer imaging, diagnosis, and therapy. J. Cell. Biochem. 114, 250-255 (2013).
25 Lee, J. W., Kim, H. J. & Heo, K. Therapeutic aptamers: developmental potential as anticancer drugs. BMB Rep. 48, 234-237 (2015).
26 Buerger, C. & Groner, B. Bifunctional recombinant proteins in cancer therapy: cell penetrating peptide aptamers as inhibitors of growth factor signaling. J. Cancer Res. Clin. 129, 669-675 (2003).
27 Kunz, C., Borghouts, C., Buerger, C. & Groner, B. Peptide aptamers with binding specificity for the intracellular domain of the ErbB2 receptor interfere with AKT signaling and sensitize breast cancer cells to Taxol. Mol. Cancer Res. 4, 983 (2006).
28 Williams, A. & Baird, L. G. DX-88 and HAE: a developmental perspective. Transfus. Apher. Sci. 29, 255-258 (2003).
29 Che, Y. J., Wu, H. W., Hung, L. Y., Liu, C. A., Chang, H. Y., Wang, K. & Lee, G. B. An integrated microfluidic system for screening of phage-displayed peptides specific to colon cancer cells and colon cancer stem cells. Biomicrofluidics 9, 054121 (2015).
30 Hung, L. Y., Wang, C. H., Che, Y. J., Fu, C. Y., Chang, H. Y., Wang, K. & Lee, G. B. Screening of aptamers specific to colorectal cancer cells and stem cells by utilizing On-chip Cell-SELEX. Sci. Rep. 5, 10326 (2015).
31 Lin, R. Z. & Chang, H. Y. Recent advances in three-dimensional multicellular spheroid culture for biomedical research. Biotechnol. J. 3, 1172-1184 (2008).
32 Ginestier, C., Hur, M. H., Charafe-Jauffret, E., Monville, F., Dutcher, J., Brown, M., Jacquemier, J., Viens, P., Kleer, C. G., Liu, S., Schott, A., Hayes, D., Birnbaum, D., Wicha, M. S. & Dontu, G. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1, 555-567 (2007).
33 Huang, E. H., Hynes, M. J., Zhang, T., Ginestier, C., Dontu, G., Appelman, H., Fields, J. Z., Wicha, M. S. & Boman, B. M. Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis. Cancer Res. 69, 3382 (2009).
34 Kalluri, R. & Zeisberg, M. Fibroblasts in cancer. Nat. Rev. Cancer 6, 392-401 (2006).
35 del Pozo, M. A., Alderson, N. B., Kiosses, W. B., Chiang, H. H., Anderson, R. G. W. & Schwartz, M. A. Integrins regulate Rac targeting by internalization of membrane domains. Science 303, 839 (2004).
36 del Pozo, M. A., Balasubramanian, N., Alderson, N. B., Kiosses, W. B., Grande-Garcia, A., Anderson, R. G. W. & Schwartz, M. A. Phospho-caveolin-1 mediates integrin-regulated membrane domain internalization. Nat. Cell Biol. 7, 901-908 (2005).
37 Kryza, D., Debordeaux, F., Azéma, L., Hassan, A., Paurelle, O., Schulz, J.,, Savona-Baron, C., Charignon, E., Bonazza, P., Taleb, J., Fernandez, P.,, Janier, M. & Toulmé, J. J. ex vivo and in vivo imaging and biodistribution of aptamers targeting the human matrix metalloprotease-9 in melanomas. PLOS ONE 11, e0149387 (2016).
38 Da Pieve, C., Blackshaw, E., Missailidis, S. & Perkins, A. C. PEGylation and biodistribution of an anti-MUC1 aptamer in MCF-7 tumor-bearing mice. Bioconjugate Chem. 23, 1377-1381 (2012).
39 Boomer, R. M., Lewis, S. D., Healy, J. M., Kurz, M., Wilson, C. & McCauley, T. G. Conjugation to polyethylene glycol polymer promotes aptamer biodistribution to healthy and inflamed tissues. Oligonucleotides 15, 183-195 (2005).
40 Puffer, B., Kreutz, C., Rieder, U., Ebert, M. O., Konrat, R. & Micura, R. 5-Fluoro pyrimidines: labels to probe DNA and RNA secondary structures by 1D (19)F NMR spectroscopy. Nucleic Acids Res. 37, 7728-7740 (2009).
41 Allured, V. S., Collier, R. J., Carroll, S. F. & McKay, D. B. Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-angstrom resolution. P. Natl. A. Sci. 83, 1320-1324 (1986).
42 Matthew, P. The role of exotoxin A in Pseudomonas disease and immunity. Rev. Infect. Dis. 5, S979-S984 (1983).
43 Michalska, M. & Wolf, P. Pseudomonas exotoxin A: optimized by evolution for effective killing. Front. Microbiol. 6, 963 (2015).
44 An, Y., Cai, Y., Guan, Y., Cai, L., Yang, Y., Feng, X. & Zheng, J. Inhibitory effect of small interfering RNA targeting insulin-like growth factor-I receptor in ovarian cancer OVCAR3 cells. Cancer Biother. Radio. 25, 545-552 (2010).
45 Bohula, E. A., Salisbury, A. J., Sohail, M., Playford, M. P., Riedemann, J., Southern, E. M. & Macaulay, V. M. The efficacy of small interfering RNAs targeted to the type 1 insulin-like growth factor receptor (IGF1R) is influenced by secondary structure in the IGF1R transcript. J. Biol. Chem. 278, 15991-15997 (2003).
46 Bhowmick, N. A., Neilson, E. G. & Moses, H. L. Stromal fibroblasts in cancer initiation and progression. Nature 432, 332-337 (2004).
47 Silzle, T., Randolph, G. J., Kreutz, M. & Kunz-Schughart, L. A. The fibroblast: sentinel cell and local immune modulator in tumor tissue. Int. J. Cancer 108, 173-180 (2004).
48 Cheng, N., Bhowmick, N. A., Chytil, A., Gorksa, A. E., Brown, K. A., Muraoka, R., Arteaga, C. L., Neilson, E. G., Hayward, S. W. & Moses, H. L. Loss of TGF-β type II receptor in fibroblasts promotes mammary carcinoma growth and invasion through upregulation of TGF-α-, MSP- and HGF-mediated signaling networks. Oncogene 24, 5053-5068 (2005).
49 Sefah, K., Meng, L., Lopez-Colon, D., Jimenez, E., Liu, C. & Tan, W. DNA aptamers as molecular probes for colorectal cancer study. PLOS ONE 5, e14269 (2010).
50 Li, Z. J. & Cho, C. H. Peptides as targeting probes against tumor vasculature for diagnosis and drug delivery. J. Transl. Med. 10, S1 (2012).