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研究生:陳俞任
論文名稱:目標化偵測Legumain酵素之螢光分子影像探針及磁振造影對比劑之開發研究
論文名稱(外文):Synthesis and Characterization of Peptide-Based Near Infrared Molecular Imaging Probe and MR Imaging Contrast Agent for Legumain Detection
指導教授:王雲銘
學位類別:碩士
校院名稱:國立交通大學
系所名稱:生物資訊及系統生物研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:92
中文關鍵詞:磁振造影光學影像
外文關鍵詞:MRIoptical imaging
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近紅外螢光比可見光能更有效率的穿透皮膚組織,至今已被運用至各種不同的生理研究;此外,磁振造影是目前臨床醫學上常使用的診斷工具,不同於其他造影技術,其對軟組織的成像具有相當的效果,但靈敏度卻稍顯不足。Legumain 酵素為一半胱氨酸蛋白酶 (cysteine protease),其在多種腫瘤上均能過度表現[1]。因此,本研究設計合成一條由胺基酸所組成的胜肽 ((L)-Leg),其對Legumain酵素具有目標性,藉由在胜肽兩端鍵結近紅外光螢光團 (CyTE777及CyTE807),以形成一近紅外光螢光探針(CyTE777-(L)-Leg-CyTE807),此近紅外光螢光探針可用於Legumain酵素活性之偵測。以高效能液相層析儀 (high-performance liquid chromatography,HPLC) 進行探針的純化後,利用質譜儀 (Mass spectrometry,MS) 鑑定其分子量為2440.92 g/mol,與實際分子量2441.17 g/mol相符。此探針上的CyTE807可吸收CyTE777的發射能量,達到抑制螢光放射之效應 (quench effect)[2,3];此探針與Legumain酵素反應後,由螢光光譜儀觀察到CyTE777原本被抑制的螢光被釋放。細胞毒性實驗結果顯示,以CyTE777-(L)-Leg-CyTE807 (10 μM) 培養下,細胞存活率達80%以上,推測若以低劑量打入動物體內,則不具有抑制細胞生長的危險性。由體外光學影像實驗中可得知,CyTE777-(L)-Leg-CyTE807與HEK-293(L) lysate進行培養後,產生明顯的螢光訊號增強,但加入HEK-293 lysate後螢光訊號無明顯增強,藉此可確認此紅外光螢光探針對Legumain酵素具有高度特異性。在體內光學影像實驗中,CyTE777-(L)-Leg-CyTE807可目標化至表現Legumain酵素 之CT-26腫瘤且提高其訊號強度。此外,本研究亦於對Legumain具有目標性之胜肽 ((L)-Leg) 之N端鍵結NBCB-TTDA,合成一對腫瘤組織中的Legumain酵素具有專一辨識功能之磁振造影對比劑(Gd-NBCB-TTDA-(L)-Leg)。在體內磁振造影實驗結果可知,Gd-NBCB-TTDA-(L)-Leg可目標化至CT-26腫瘤且提高其訊號強度,而增加腫瘤位置之辨識度。綜合以上結果,本研究所設計合成之光學影像探針 (CyTE777-(L)-Leg-CyTE807) 及磁振造影對比劑(Gd-NBCB-TTDA-(L)-Leg) 均對Legumain酵素具有高度特異性,可應用於Legumain表現之腫瘤進行診斷及追蹤。
Near-infrared fluorescence has been applied to image various biological events in vivo, because of higher efficiency of penetration than that of visible radiations. Besides, magnetic resonance imaging (MRI) has become the leading tool for imaging and ability to resolve different soft tissues. MRI has not been effectively used for molecular imaging because of its low sensitivity.In this study, an 8-amino acids peptide substrate was designed and synthesized for specific recognition of legumain enzyme. A near-infrared fluorochrome serving as the optical imager can be used for in vivo imaging of enzyme activity. So we developed an optical probe (CyTE777-(L)-Leg-CyTE807) that consisted of a recognized sequence that was flanked by a fluorophore-quencher pair (CyTE777 and CyTE807). HPLC was employed to purify the optical probe, and mass spectrometer was used to appraise the result. Its molecular weight is 2440.92 g/mole, and intrinsical molecular weight is 2441.17 g/mole. Once exposed to legumain enzyme, the peptide was cleaved, resulting in separation of the fluorophore pair and signal generation. When CyTE777-(L)-Leg-CyTE807 was cleaved by legumain enzyme, the CyTE777 emission (840 nm) was released. The cytotoxicity experiment showed that cell incubated with the probe, the cell viability maintained up to 80%. In vitro studies showed CyTE777-(L)-Leg-CyTE807 incubated with HEK-293(L) cell lysate, the signal intensity significantly enhanced. However, the probe incubated with control legumain enzyme (HEK-293), the signal intensity has no obvious signal enhancement. In vivo studies demonstrated that this probe could specifically recognize CT-26 tumor. On the other hand, we also designed and synthesized a novel contrast agent (CA), Gd-NBCB-TTDA-(L)-Leg, to image the activity of legumain in a tumor noninvasively using MR imaging. The in vivo studies demonstrated that, the contrast agent would be specifically recognized by legumain enzyme around the tumor, which results in significant tumor contrast enhancement at a dose of 0.1 mmol Gd / kg for at least 60 min in mice bearing CT-26 tumor for MR images. As a conclusion, there results indicate that the optical probe and the contrast agent have low cytotoxicity, and specific tumor localization. These optical probe and contrast agent have a potential for cancer molecular imaging with optical imaging and MR imaging.
誌謝 I
摘要 II
Abstract IV
目錄 VI
圖目錄 X
一、前言 1
1.1 分子影像 (Molecular imaging) 1
1.2 光學影像顯影技術 (Optical imaging) 3
1.2.1 光學影像探針 (Optical imaging probe) 3
1.3 磁振造影技術 (Magnetic resonance imaging,MRI) 10
1.3.1 磁振造影原理 11
1.3.2磁振造影對比劑 (MRI contrast agent) 12
1.3.3磁振造影對比劑之種類 12
1.3.4 順磁性磁振造影對比劑之研究現況 13
1.4 研究目的與動機 26
二、 儀器與試藥 28
2.1 儀器 (Instrumentation) 28
2.2 試藥 (Reagents) 30
三、實驗方法 33
3.1胜肽受質之合成與溶劑製備 33
3.1.1偶合劑之製備 33
3.1.2活化劑之製備 33
3.1.3 封端劑 (capping agent) 之製備 33
3.1.4 Kaiser test所需之溶劑製備 34
3.1.5切除試劑及去保護基之溶劑製備 34
3.1.6 固相胜肽合成法 35
3.1.7 實驗組胜肽 ((L)-Leg) 及對照組胜肽 ((D)-Leg) 之合成 37
3.2螢光染料CyTE777之合成 38
3.3螢光染料CyTE807之合成 39
3.4螢光染料CyTE777 與樹脂的偶合反應 40
3.5螢光染料CyTE807與樹脂的偶合及切割 41
3.6 NBCB-NCS-5est (7) 之製備 44
3.6.1 C-(1-aminomethyl-cyclobutyl)-methylamine (CB-amine, 1)之製備 44
3.6.2 p-nitrophenylalanine methylester (NBPDA, 2)之製備 45
3.6.3 2-amino-N-(1-aminomethyl-cyclobutyl)-3-(4-nitrophenyl)- propionamide (NBCB, 3)之製備 45
3.6.4 NBCB-NO2-5est (5) 之製備 46
3.6.5 NBCB-NH2-5est (6) 之製備 47
3.6.6 NBCB-COOH-5est (7) 之製備 48
3.7 NBCB-TTDA-Leg之合成 49
3.7.1 NBCB-TTDA-Leg之合成 49
3.7.2 切除resin與保護基 50
3.7.3 NBCB-TTDA-Leg純化與鑑定 50
3.8 NBCB-TTDA-Leg釓及銪金屬錯合物之合成 51
3.9 細胞株及培養 51
3.10 CyTE777-(L)-Leg-CyTE807細胞毒性實驗 51
3.11 體外光學影像實驗 52
3.12 體內光學影像實驗 52
3.13 動物體分佈實驗 53
3.14體外磁振造影實驗 53
3.15 體內磁振造影實驗 54
四、結果與討論 55
4.1 胜肽合成機制之探討 55
4.1.1 Kaiser test 55
4.1.2 Resin的去保護 56
4.1.3 胺基酸的活化 57
4.1.4 胺基酸與resin的偶合 57
4.1.5 Capping 58
4.1.6 去除離胺酸之ivDde保護基 59
4.1.7 胺基酸上tert-Bu(tert-butyl)與Boc保護基的切除 60
4.2 光學影像探針之特性探討 61
4.2.1螢光染料CyTE777及CyTE807的純化 61
4.2.2螢光染料CyTE777及CyTE807的鑑定 63
4.2.3 實驗組胜肽受質及對照組胜肽受質之鑑定 64
4.2.4 CyTE777-(L)-CyTE807的純化及鑑定 65
4.3 螢光染料CyTE777之螢光測試 67
4.4 螢光染料CyTE807之螢光測試 68
4.5 CyTE777-(L)-Leg-CyTE807之螢光測試 70
4.6 Legumain酵素誘導胜肽切割 70
4.7細胞毒性之探討 72
4.8 體外光學影像研究 73
4.9 體內光學影像研究 74
4.10 動物生物體分佈研究 77
4.11 NBCB-COOH-5est合成反應探討 80
4.12 切割型磁振造影對比劑Gd-NBCB-TTDA-Leg之探討 80
4.12.1製備NBCB-TTDA-Leg合成反應機制 80
4.12.2 NBCB-TTDA-Leg純化及鑑定 82
4.12.3 Gd-NBCB-TTDA-Leg純化及鑑定 83
4.12.4 體外磁振造影研究 85
4.12.5體內磁振造影研究 86
五、結論 88
六、參考文獻 89
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