進階搜尋


   電子論文尚未授權公開,紙本請查館藏目錄
(※如查詢不到或館藏狀況顯示「閉架不公開」,表示該本論文不在書庫,無法取用。)
系統識別號 U0026-1908201416475800
論文名稱(中文) Galectin-1 aptamers應用於纖維母細胞的調控
論文名稱(英文) Application of Galectin-1 aptamers in fibroblast activity
校院名稱 成功大學
系所名稱(中) 口腔醫學研究所
系所名稱(英) Institute of Oral Medicine
學年度 102
學期 2
出版年 103
研究生(中文) 楊東錦
研究生(英文) Tung-Chin Yang
學號 T46014010
學位類別 碩士
語文別 英文
論文頁數 47頁
口試委員 指導教授-陳玉玲
口試委員-洪澤民
口試委員-謝達斌
口試委員-袁國
中文關鍵字 半乳糖凝集素-1  纖維母細胞  適體 
英文關鍵字 Galectin-1  Fibroblast  aptamers 
學科別分類
中文摘要 適體是由去氧核糖核酸或核糖核酸所組成的短序列,可以透過本身的二級結構與特定的目標蛋白序列結合並且抑制該蛋白的功能表現。半乳醣凝集素-1含有碳水化合物的辨識區,在我們先前的研究中證明半乳醣凝集素-1在癌細胞貼附,移行,以及癌化進程等病理現象中扮演重要的調控角色,另外我們也在先前研究中發現,半乳醣凝集素-1會影響正常細胞的生理活性,像是促進纖維母細胞增生與活化,纖維母細胞的活化不論是在生理或是病理上都扮演著極為重要的角色,像是傷口癒合或肝纖維化。我們先前已篩選得到半乳醣凝集素-1的DNA適體AP27, AP39, 和AP74,並證實口腔癌細胞貼附能力會隨著加入AP-27, AP-39的濃度升高而降低。在這份研究中我們發現半乳醣凝集素-1適體可以有效地抑制半乳醣凝集素-1誘導的纖維母細胞活化、細胞增生和爬行能力,在纖維母細胞上這些適體可抑制半乳醣凝集素-1所促進的Smad3磷酸化。另外在肺癌細胞中,我們發現半乳醣凝集素-1適體也可有效抑制半乳醣凝集素-1所增強肺癌細胞的爬行能力,而在肺癌細胞上這些適體可抑制半乳醣凝集素-1所促進的Akt/mTOR磷酸化。本篇論文中,我們進一步找出半乳醣凝集素-1適體對於纖維母細胞以及肺癌細胞可能透過不同調控機制而抑制其細胞爬行與增生能力。未來,透過適體微陣列晶片期望找出半乳醣凝集素適體的有效片段,優化半乳醣凝集素適體的效率也降低製作的成本。希望可以利用半乳醣凝集素-1適體為基礎而發展出獨特的療法來治療與纖維母細胞活化相關的疾病和預防癌細胞的轉移。
英文摘要 Aptamers are short DNA or RNA sequence that can bind to specific sites on proteins through its secondary conformation and structure. The binding of the aptamer to the protein can result in blockage of specific protein functions. Previous studies from our group have identified aptamers AP-27, AP-39, and AP-74 which possess high binding affinity for galectin-1 (Gal-1), a member of galectin family that has a carbohydrate recognition domain (CRD) to bind to β-galactosides. Gal-1 plays a critical role in the regulation of cell adhesion, migration, tumorigenesis and promoting cancer cells metastasis. Furthermore, we also found that Gal-1 influences the several biological functions of normal cells such as promoting fibroblast proliferation and activation. Activation of fibroblasts plays important role in several physiological and pathological processes such as wound healing and liver fibrosis. We have demonstrated that AP-27 and AP-39 dose-dependently inhibit Gal-1-induced oral cancer cell adhesion. In this study, we found that the aptamers efficiently rescued Gal-1-activated fibroblast migration and proliferation. These aptamers, especially AP-74, decreased Gal-1-induced phosphorylation of Smad3 in fibroblasts. On the other hand, we also found that these aptamers inhibited Gal-1-induced lung cancer cell migration and might through inhibiting Akt/mTOR signaling pathway. In the future, the Gal-1 aptamers will be optimized by screening a custom aptamer array chip to search out the effective region of Gal-1 aptamers for costing down and further applications. The development of Gal-1 aptamer-based therapeutics may provide unique clinical opportunities for diseases associated with fibroblast activation, and preventing cancer cell metastasis.
論文目次 摘要 I
Abstract II
Acknowledgment IV
Contents VI
Introduction 1
The selection and properties of aptamers 1
Gal-1 in cell migration 5
Gal-1 in lung cancer 6
Fibroblast and activated fibroblast 6
Lung cancer cell 7
Rationale and specific aims 9
Materials and methods 11
Primary culture of HGF and CAF 11
Cell culture 11
Results 21
Gal-1 promotes fibroblast activation and migration 21
Gal-1 aptamers rescues gal-1-induced fibroblast migration and proliferation 21
Gal-1 aptamers reduced Gal-1-induced Smad3 phosphorylation in HGFs 22
Gal-1 aptamers reduce gal-1-promoted lung cancer cells migration 23
Discussion 25
Differences of galectin-1 aptamers blocking efficiency 25
NRP-1 as galectin-1 receptor regulates in cells protein expression. 25
Applications of gal-1 aptamers in therapeutics 26
Conclusion 28
References 30
Figures 39
Figure 1. Gal-1 dose-dependently promotes human gingival fibroblast (HGF) migration. 39
Figure 2. Gal-1 activates fibroblasts into activated-fibroblasts. 40
Figure 3. Gal-1 aptamers can rescue the tendency that Gal-1 activating cell migration. 41
Figure 4. Gal-1 aptamers have no toxicity in HGF and CAF. 42
Figure 5. Gal-1 aptamer co-treated with Gal-1 can reduce HGF cell viability. 43
Figure 6. Gal-1 aptamers reduced Gal-1-induced Smad3 phosphorylation in HGF. 44
Figure 7. Gal-1 dose-dependently enhances CL 1-0 migration ability. 45
Figure 8. AP-27 and AP-74 effectively reduce Gal-1-activated CL 1-0 migration. 46
Figure 9. Gal-1 aptamers block Gal-1 activating AKT/mTOR signaling pathway in CL1-0. 47
參考文獻 Bala, J., Bhaskar, A., Varshney, A., Singh, A.K., Dey, S., and Yadava, P. (2011). In vitro selected RNA aptamer recognizing glutathione induces ROS mediated apoptosis in the human breast cancer cell line MCF 7. RNA Biology 8, 101-111.
Barsky, S.H., Green, W.R., Grotendorst, G.R., and Liotta, L.A. (1984). Desmoplastic breast carcinoma as a source of human myofibroblasts. The American Journal of Pathology 115, 329-333.
Belanis L, Plowman SJ, Rotblat B, Hancock JF, Kloog Y. (2008). Galectin-1 is a novel structural component and a major regulator of H-ras nanoclus- ters. Molecule Biology Cell 19, 1404–14.
Breathnach, O. S., Freidlin, B., Conley, B., Green, M. R., Johnson, D. H., Gandara, D. R., O'Connell, M., Shepherd, F. A., and Johnson, B. E. (2001). Twenty-two years of phase III trials for patients with advanced non-small-cell lung cancer: sobering results. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 19, 1734-1742.
Camby I, Belot N, Rorive S, et al. (2011). Galectins are differentially expressed in supratentorial pilocytic astrocytomas, astrocytomas, anaplastic astrocytomas and glioblastomas, and significantly modulate tumor astrocyte migration. Brain Pathology 11, 12-26.
Camby I, Le Mercier M, Lefranc F, Kiss R. (2006). Galectin-1: a small protein with major functions. Glycobiology 16, 137R–57R.
Camby I, Mercier ML, Lefranc F, Kiss R. (2006). Galectin-1: A small protein with major functions. Glycobiology 16, 137-57.
Camby, I., Decaestecker, C., Lefranc, F., Kaltner, H., Gabius, H.J., and Kiss, R. (2005). Galectin-1 knocking down in human U87 glioblastoma cells alters their gene expression pattern. Biochem Biophys Res Commun 335, 27-35.
Camby, I., Le Mercier, M., Lefranc, F., and Kiss, R. (2006). Galectin-1: a small protein with major functions. Glycobiology 16, 137-157.
Chang, Y.C., Kao, W.C., Wang, W.Y., Yang, R.B., and Peck, K. (2009). Identification and characterization of oligonucleotides that inhibit Toll-like receptor 2-associated immune responses. Faseb J 23, 3078-3088.
Chen YR, Juan HF, Huang HC, et al. (2006). Quantitative proteomic and genomic profiling reveals metastasis-related protein expression patterns in gastric cancer cells. J Proteome Res 5, 2727-42.
Cox JC, Rudolph P, Ellington AD. 1998. Automated RNA selection. Biotechnol. Prog 14, 845-850.
Crino, L., Weder, W., van Meerbeeck, J., and Felip, E. (2010). Early stage and locally advanced (non-metastatic) non-small-cell lung cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO 21 Suppl 5, v103-115.
De Wever, O., Demetter, P., Mareel, M., and Bracke, M. (2008). Stromal myofibroblasts are drivers of invasive cancer growth. Int J Cancer 123, 2229-2238.
Dimanche-Boitrel, M.T., Vakaet, L., Jr., Pujuguet, P., Chauffert, B., Martin, M.S., Hammann, A., Van Roy, F., Mareel, M., and Martin, F. (1994). In vivo and in vitro invasiveness of a rat colon-cancer cell line maintaining E-cadherin expression: an enhancing role of tumor-associated myofibroblasts. Int J Cancer 56, 512-521.
Eaton, B.E., Gold, L., Hicke, B.J., Janjic, N., Jucker, F.M., Sebesta, D.P., Tarasow, T.M., Willis, M.C., and Zichi, D.A. (1997). Post-SELEX combinatorial optimization of aptamers. Bioorg Med Chem 5, 1087-1096.
Ellerhorst, J., Nguyen, T., Cooper, D.N., Estrov, Y., Lotan, D., and Lotan, R. (1999a). Induction of differentiation and apoptosis in the prostate cancer cell line LNCaP by sodium butyrate and galectin-1. Int J Oncol 14, 225-232.
Ellerhorst, J., Nguyen, T., Cooper, D.N., Lotan, D., and Lotan, R. (1999b). Differential expression of endogenous galectin-1 and galectin-3 in human prostate cancer cell lines and effects of overexpressing galectin-1 on cell phenotype. Int J Oncol 14, 217-224.
Ellington AD, Szostak JW. 1990. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–22.
Ferreira, C.S., Matthews, C.S., and Missailidis, S. (2006). DNA aptamers that bind to MUC1 tumour marker: design and characterization of MUC1-binding single-stranded DNA aptamers. Tumour Biol 27, 289-301.
Fischer C, Sanchez-Ruderisch H, Welzel M, Wiedenmann B, Sakai T, Andre S, et al. (2005). Galectin-1 interacts with the a5b1 fibronectin receptor to restrict carcinoma cell growth via induction of p21 and p27. J Biol Chem 280:37266–77.
Goldring, K., Jones, G. E. and Watt, D. J. (2000). A factor implicated in the myogenic conversion of non-muscle cells derived from the mouse dermis. Cell Transplantation 9, 519-529.
Guizhi Zhu, Jing Zheng, Erqun Song, Michael Donovan, Kejing Zhang, Chen Liu, Weihong Tan (2012). Self-assembled, aptamer-tethered DNA nanotrains for targeted transport of molecular drugs in cancer theranostics 7998–8003.
Hammerman, P. S. , Fox, C. J. & Thompson, C. B. (2004) Beginnings of a signal-transduction pathway for bioenergetic control of cell survival. Trends Biochem. Sci. 29, 586–592.
Hittelet, A., Legendre, H., Nagy, N., Bronckart, Y., Pector, J.C., Salmon, I., Yeaton, P., Gabius, H.J., Kiss, R., and Camby, I. (2003). Upregulation of galectins-1 and -3 in human colon cancer and their role in regulating cell migration. Int J Cancer 103, 370-379.
Holohan, C., Van Schaeybroeck, S., Longley, D. B., and Johnston, P. G. (2013). Cancer drug resistance: an evolving paradigm. Nature reviews Cancer 13, 714-726.
Hsieh, S.H., Ying, N.W., Wu, M.H., Chiang, W.F., Hsu, C.L., Wong, T.Y., Jin, Y.T., Hong, T.M., and Chen, Y.L. (2008). Galectin-1, a novel ligand of neuropilin-1, activates VEGFR-2 signaling and modulates the migration of vascular endothelial cells. Oncogene.
Jiang, L., Suri, A.K., Fiala, R., and Patel, D.J. (1997). Saccharide-RNA recognition in an aminoglycoside antibiotic-RNA aptamer complex. Chem Biol 4, 35-50.
Jung EJ, Moon HG, Cho BI, et al. (2007). Galectin-1 expression in cancer-associated stromal cells correlates tumor invasiveness and tumor progression in breast cancer. Int J Cancer 120(11), 2331-8.
Jung EJ, Moon HG, Cho BI, Jeong CY, Joo YT, Lee YJ, et al. (2007). Galectin-1 expression in cancer-associated stromal cells correlates tumor inva- siveness and tumor progression in breast cancer. Int J Cancer 120, 2331–8.
Jung, E.J., Moon, H.G., Cho, B.I., Jeong, C.Y., Joo, Y.T., Lee, Y.J., Hong, S.C., Choi, S.K., Ha, W.S., Kim, J.W., et al. (2007). Galectin-1 expression in cancer-associated stromal cells correlates tumor invasiveness and tumor progression in breast cancer. Int J Cancer 120, 2331-2338.
Kalluri, R., and Zeisberg, M. (2006). Fibroblasts in cancer. Nat Rev Cancer 6, 392-401.
Kawashiri, S., Tanaka, A., Noguchi, N., Hase, T., Nakaya, H., Ohara, T., Kato, K., and Yamamoto, E. (2009). Significance of stromal desmoplasia and myofibroblast appearance at the invasive front in squamous cell carcinoma of the oral cavity. Head Neck 31, 1346-1353.
Kovacs-Solyom F, Blasko A, Fajka-Boja R, Katona RL, Vegh L, Novak J, et al. (2010). Mechanism of tumor cell-induced T-cell apoptosis mediated by galectin-1. Immunol Lett 127, 108–18.
Kreunin P, Yoo C, Urquidi V,
Lubman DM, Goodison S. (2007). Proteomic profiling identifies breast tumor metastasis-associated factors in an isogenic model. Proteomics 7(2), 299-312.
Lahm H, Andre S, Hoeflich A, et al. (2001). Comprehensive galectin fingerprinting in
a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures. J Cancer Res Clin Oncol 127(6), 375-86.
Lee, J.H., Canny, M.D., De Erkenez, A., Krilleke, D., Ng, Y.S., Shima, D.T., Pardi, A., and Jucker, F. (2005). A therapeutic aptamer inhibits angiogenesis by specifically targeting the heparin binding domain of VEGF165. Proc Natl Acad Sci U S A 102, 18902-18907.
Leffler H, Carlsson S, Hedlund M, Qian Y, Poirier F (2004) Introduction to galectins. Glycoconj J 19, 433–440.
Liu FT, Rabinovich GA. (2005). Galectins as modulators of tumour progression.
Nat Rev Cancer 5(1), 29-41.
Lorsch, J.R., and Szostak, J.W. (1994). In vitro selection of RNA aptamers specific for cyanocobalamin. Biochemistry 33, 973-982.
Lotan, R., Belloni, P.N., Tressler, R.J., Lotan, D., Xu, X.C., and Nicolson, G.L. (1994). Expression of galectins on microvessel endothelial cells and their involvement in tumour cell adhesion. Glycoconj J 11, 462-468.
Mi, J., Zhang, X., Rabbani, Z.N., Liu, Y., Su, Z., Vujaskovic, Z., Kontos, C.D., Sullenger, B.A., and Clary, B.M. (2006). H1 RNA polymerase III promoter-driven expression of an RNA aptamer leads to high-level inhibition of intracellular protein activity. Nucleic Acids Res 34, 3577-3584.
Nishikawa, F., Kakiuchi, N., Funaji, K., Fukuda, K., Sekiya, S., and Nishikawa, S. (2003). Inhibition of HCV NS3 protease by RNA aptamers in cells. Nucleic Acids Res 31, 1935-1943.
Ohannesian, D.W., Lotan, D., and Lotan, R. (1994). Concomitant increases in galectin-1 and its glycoconjugate ligands (carcinoembryonic antigen, lamp-1, and lamp-2) in cultured human colon carcinoma cells by sodium butyrate. Cancer Research 54, 5992-6000.
Ohuchida, K., Mizumoto, K., Murakami, M., Qian, L.W., Sato, N., Nagai, E., Matsumoto, K., Nakamura, T., and Tanaka, M. (2004). Radiation to stromal fibroblasts increases invasiveness of pancreatic cancer cells through tumor-stromal interactions. Cancer Research 64, 3215-3222.
Orimo, A., Gupta, P.B., Sgroi, D.C., Arenzana-Seisdedos, F., Delaunay, T., Naeem, R., Carey, V.J., Richardson, A.L., and Weinberg, R.A. (2005). Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 121, 335-348.
Paz A, Haklai R, Elad-Sfadia G, Ballan E, Kloog Y. (2001). Galectin-1 binds oncogenic H-Ras to mediate Ras membrane anchorage and cell transformation. Oncogene 20, 7486-93.
Prior IA, Muncke C, Parton RG, Hancock JF. (2003). Direct visualization of Ras proteins in spatially distinct
cell surface microdomains. J Cell Biol 160, 165-70.
Puchades M, Nilsson CL, Emmett MR,et al. (2007). Proteomic investigation of glioblastoma cell lines treated with wild-type p53 and cytotoxic chemotherapy demonstrates an association between galectin-1 and p53 expression. J Proteome Res 6, 869-75.
Rabinovich GA, Ilarregui JM. (2009). Conveying glycan information into T-cell homeostatic programs a challenging role for galectin-1 in inflammatory and tumor microenvironments. Immunol Rev 230, 144–59.
Rubinstein N, Alvarez M, Zwirner NW,
et al. (2004). Targeted inhibition of galectin-1 gene expression in tumor cells results in heightened T cell-mediated rejection; a potential mechanism of tumor-immune privilege. Cancer Cell 5, 241-51.
Sanford, G. L. and Harris-Hooker, S. (1990). Stimulation of vascular cell proliferation by beta–galactoside specific lectins. FASEB J. 4, 2912-2918.
Seelenmeyer, C., Stegmayer, C., and Nickel, W. (2008). Unconventional secretion of fibroblast growth factor 2 and galectin-1 does not require shedding of plasma membrane-derived vesicles. FEBS Lett 582, 1362-1368.
Spano, D., Russo, R., Di Maso, V., Rosso, N., Terracciano, L.M., Roncalli, M., Tornillo, L., Capasso, M., Tiribelli, C., and Iolascon, A. (2010). Galectin-1 and its involvement in hepatocellular carcinoma aggressiveness. Mol Med 16, 102-115.
Szoke T, Kayser K, Baumhakel JD, Trojan I, Furak J, Tiszlavicz L, et al. (2005). Prognostic significance of endogenous adhesion/growth-regulatory lectins in lung cancer. Oncology 69, 167–74.
Tavitian, B. (2003). In vivo imaging with oligonucleotides for diagnosis and drug development. Gut 52 Suppl 4, iv40-47.

Thiel, K.W., and Giangrande, P.H. (2010). Intracellular delivery of RNA-based therapeutics using aptamers. Ther Deliv 1, 849-861.
Tuerk C, Gold L. (1990). Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–10.
Van Den Brule F, Califice S, Castronovo V. (2004). Expression of galectins in cancer: a critical review. Glycoconj J 19, 537-42.
van den Brule F, Califice S, Garnier F, Fernandez PL, Berchuck A, Castronovo V. (2003). Galectin-1 accumulation in the ovary carcinoma peri- tumoral stroma is induced by ovary carcinoma cells and affects both cancer cell proliferation and adhesion to laminin-1 and fibronectin. Lab Invest 83, 377–86.
Van Den Brule FA, Waltregny D, Castronovo V. (2001). Increased expression of galectin-1 in carcinoma-associated stroma predicts poor outcome in prostate carcinoma patients. J Pathol 193, 80-7.
van den Brule, F., Califice, S., Garnier, F., Fernandez, P.L., Berchuck, A., and Castronovo, V. (2003). Galectin-1 accumulation in the ovary carcinoma peritumoral stroma is induced by ovary carcinoma cells and affects both cancer cell proliferation and adhesion to laminin-1 and fibronectin. Laboratory Investigation 83, 377-386.
van den Brule, F.A., Buicu, C., Baldet, M., Sobel, M.E., Cooper, D.N., Marschal, P., and Castronovo, V. (1995). Galectin-1 modulates human melanoma cell adhesion to laminin. Biochem Biophys Res Commun 209, 760-767.
Vespa, G. N., Lewis, L. A., Kozak, K. R., Moran, M., Nguyen, J. T., Baum, L. G. and Miceli, L. C. (1999). Galectin-1 specifically modulates TCR signals to enhance TCR apoptosis but inhibit IL-2 production and proliferation. J. Immunol. 162, 799-806.
Walzel, H., Schulz, U., Neels, P. and Brock, J. (1999). Galectin-1, a natural ligand for the receptor-type protein tyrosine phosphatase CD45. Immunol. Lett. 67, 193-202.
Weigelt, B., Peterse, J. L., and van 't Veer, L. J. (2005). Breast cancer metastasis: markers and models. Nature reviews Cancer 5, 591-602.
Wells, V. and Malluchi, L. (1991). Identification of an autocrine negative growth factor: murine beta-galactoside- binding protein is a cytostatic factor and cell growth regulator. Cell 64, 91-97.
Wittekind, C., and Neid, M. (2005). Cancer invasion and metastasis. Oncology 69 Suppl 1, 14-16.
Wu MH, Hong HC, Hong TM, Chiang WF, Jin YT, Chen YL. (2011). Targeting galectin-1 in carcinoma-associated fibroblasts inhibits oral squamous cell carcinoma metastasis by downregulating MCP-1/CCL2 expres- sion. Clin Cancer Res 17, 1306–16.
Wu MH, Hong TM, Cheng HW, Pan SH, Liang YR, Hong HC, et al. (2009). Galectin-1-mediated tumor invasion and metastasis, up-regulated matrix metalloproteinase expression, and reorganized actin cyto- skeletons. Mol Cancer Res 7, 311–8.
Yamaoka, K., Ohno, S., Kawasaki, H. and Suzuki, K. (1991). Overexpression of a beta-galactoside binding protein causes transformation of BALB3T3 fibroblast cells. Biochem. Biophys. Res. Commun 179, 272- 279.
Yu, M.K., Kim, D., Lee, I.H., So, J.S., Jeong, Y.Y., and Jon, S. (2011). Image-Guided Prostate Cancer Therapy Using Aptamer-Functionalized Thermally Cross-Linked Superparamagnetic Iron Oxide Nanoparticles. Small.
Zhang, Y., Lu, H., Dazin, P., and Kapila, Y. (2004). Squamous cell carcinoma cell aggregates escape suspension-induced, p53-mediated anoikis: fibronectin and integrin alphav mediate survival signals through focal adhesion kinase. The Journal of Biological Chemistry 279, 48342-48349.
論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2024-12-31起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw