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
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 
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
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