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系統識別號 U0026-2807201716162700
論文名稱(中文) Neuropilin-1短片段適體篩選及應用
論文名稱(英文) The screening and application of short aptamers against neuropilin-1
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 105
學期 2
出版年 106
研究生(中文) 吳宜欣
研究生(英文) Yi-Hsin Wu
學號 T46044031
學位類別 碩士
語文別 中文
論文頁數 58頁
口試委員 指導教授-陳玉玲
口試委員-洪澤民
口試委員-蘇文彬
口試委員-吳梨華
中文關鍵字 神經纖毛蛋白-1  適體  血管新生 
英文關鍵字 Neuropilin1  aptamer  angiogenesis 
學科別分類
中文摘要 適體為單股的DNA或RNA可以透過形成二級結構或三級結構而與目標分子物進行結合,適體模擬了抗體的特性,但比起抗體有更強的專一性及親和性,在臨床上可以用於與致癌基因的結合而達到治療及檢測的用途。Neuropilin-1 (NRP1)是一個細胞膜上的蛋白質,可以促進癌症的形成,包括腫瘤的增生、腫瘤細胞的侵襲性、轉移性及血管新生。由於他細胞質內的結構較短,大多被認為扮演VEGFA165, SEMA3A及其他配體共受體的角色。在非小細胞肺癌患者中發現NRP1的高表現會與病人的低存活率及高腫瘤復發能力相關,在實驗室先前的研究中,已經有針對NRP1蛋白質篩選出專一性高的適體(AP5, AP15, 和 AP37),雖然這三條適體對於NRP1都有高親和性及專一性的結合,但合成70-80的核甘酸序列用於未來活體實驗上成本仍然是過高,因此我們希望能在這篇研究中優化及縮短與NRP1具有專一性的適體。我們認為縮短的NRP1專一性的適體透過與NRP1結合,可應用於癌症的診斷及治療。本研究我們透過AP5、AP15和AP37的序列製作了一個短適體的微陣列,透過與NRP1的結合,得到幾個與NRP1具有高親和性的短片段適體。第一,我們把有表現NRP1的細胞株與短片段適體進行親和性及專一性的能力分析,證實短片段適體確實會與NRP1有專一性的結合。第二,檢測短片段適體在各種生物功能上的影響,發現短片段的適體不具有細胞毒性,且Ap5-447短片段適體會透過減少絲狀偽足的形成而抑制肺癌細胞的遷移能力,也有抑制細胞侵襲的能力。第三,在3D結構預測中也發現Nrp1專一性適體會纏繞在Nrp1 domain上,透過影響Nrp1蛋白的結構或配體與Nrp1的結合而影響下游訊路徑。第四,Ap5-447適體在動物實驗中也會透過抑制血管新生能力而抑制小鼠腫瘤生長速率,另一方面,我們也在動物體中以同位素的方式去追蹤適體進入體內後的動向,Ap5-447在24小時後能夠專一的貼附在腫瘤微轉移的位置。這個研究表明了NRP1的短片段適體在未來可能會開發成為肺癌的有用的診斷工具及治療策略。
英文摘要 Neuropilin-1 (NRP1) can facilitate cancer progression, including tumor growth, invasion, metastasis and angiogenesis. Non-small cell lung cancer patients with aberrant upregulation of NRP1 have poor disease-free and overall survival rate. In this study, we selected the truncated NRP1-specific aptamers, we found Ap5-447 aptamer inhibited migratory by decreasing filopodia formation, decreasing invasive ability. Intratumoral injection of Ap5-447 aptamer induced a significant decrease in the growth of CL 1-5 xenograft tumors by inhibit angiogenesis. This study revealed that NRP1-specific aptamers might be developed useful diagnostic and therapeutic strategies to lung cancer in the future.
論文目次 摘要 i
Extended Abstract iii
致謝 v
目錄 vi
英文縮寫檢索表 viii
第一章 緒論 1
癌症的治療策略 1
適體 (Aptamer) 1
適體在藥物發展中的應用 2
癌症 3
肺癌 3
Neuropilin-1 (NRP1) 5
NRP1在癌症中的影響 6
研究動機 7
第二章 材料與方法 8
一、細胞培養(cell culture) 8
二、細胞蛋白質表現分析(protein expression assay) 10
2.1 細胞蛋白質樣品收集 10
2.2 蛋白質定量 11
2.3 西方墨點法 11
三、細胞功能分析(cell function assay) 15
3.1 細胞毒性分析 15
3.2 細胞遷移分析 15
3.3 細胞侵襲分析 16
3.4 體外血管新生分析 16
四、免疫螢光染色(immunofluorescence) 17
五、小鼠腫瘤動物模式建立 18
5.1 皮下注射 (Subcutaneous injection assay) 18
5.2 免疫組織化學染色法(immunohistochemistry, IHC) 19
六、動物體內同位素追蹤 21
6.1 Nrp1-aptamer-DTPA之修飾 21
6.2 111In-DTPA-aptamer標幟 21
七、統計方式 22
第三章 實驗結果 23
一、以微陣列篩選短片段Nrp1專一性適體 23
二、短片段Nrp1專一性適體與Nrp1有專一性結合能力 23
三、短片段Nrp1專一性適體 (Ap5-447) 在肺癌細胞CL1-5中不具細胞毒性且能抑制細胞遷移及細胞侵襲能力 24
四、短片段Nrp1專一性適體 (Ap5-447) 在肺癌細胞A549中不具細胞毒性且能抑制細胞遷移及細胞侵襲能力 25
五、短片段Nrp1專一性適體 (Ap5-447) 在臍靜脈內皮細胞(I-Huvec)中能抑制細胞遷移能力 26
六、Ap5-447透過減少絲狀偽足的形成而抑制細胞遷移能力 26
七、以3D結構預測適體與Nrp1蛋白的結合位置 27
八、Nrp1-Ap5-447在動物體中會抑制腫瘤的生長 27
九、Nrp1-Ap5-447透過降低血管新生的作用而抑制腫瘤生長 28
十、在小鼠體內以放射設性同位素標記適體追蹤適體 29
第四章 討論 30
第五章 結論 34
第六章 參考文獻 35
第七章 圖 41
圖一:Nrp1專一性適體序列分析比對 41
圖二:短片段Nrp1適體與Nrp1有專一性結合能力 43
圖三:短片段Nrp1專一性適體 (Nrp1-Ap5-447) 在肺癌細胞CL1-5中不具細胞毒性且能抑制細胞遷移及細胞侵襲能力 45
圖四:短片段Nrp1專一性適體 (Nrp1-Ap5-447) 在肺癌細胞A549中不具細胞毒性且能抑制細胞遷移及細胞侵襲能力 47
圖五:短片段Nrp1專一性適體 (Nrp1-Ap5-447)能抑制血管內皮細胞的遷移能力 49
圖六:Nrp1-Ap5-447透過減少絲狀偽足的形成而抑制細胞遷移能力 50
圖七:以3D結構預測適體與Nrp1蛋白的結合位置 52
圖八:Nrp1-Ap5-447在動物體中會抑制腫瘤的生長 53
圖九:Nrp1-Ap5-447透過降低血管新生的作用而抑制腫瘤生長 55
圖十:在小鼠體內以放射設性同位素標記適體追蹤適體 57
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