臺灣博碩士論文加值系統

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。因此本研究設計合成一條由8個胺基酸所組成的胜肽，其對Legumain酵素具有目標性，且在多種腫瘤上都有過度表現。並在胜肽N端鍵結NBCB-TTDA，形成對腫瘤組織中的Legumain具有專一辨識功能的磁振造影對比劑Gd-NBCB-TTDA-Legumain。在20 MHz、37.0 ± 0.1 °C下，Gd-NBCB-TTDA-Legumain所求得弛緩率(r1)為4.94 mM-1s-1。在T1弛緩時間變化量的實驗中發現，當有存在3T3-Legumain cell lysate時，T1約上升20 %。在細胞毒性實驗中發現，在高濃度(1 mM) Gd-NBCB-TTDA-Legumain培養下，細胞存活率仍達85 %以上，推測若以低劑量打入動物體內不具有抑制細胞生長的危險性。在體內(in vivo)實驗研究中，Gd-NBCB-TTDA-Legumain可目標化至CT-26腫瘤且提高其訊號強度，而使腫瘤位置容易辨別。結果顯示此磁振造影對比劑有潛力對有Legumain表現之腫瘤進行診斷及追蹤。

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, we designed and synthesized a novel contrast agent (CA),
Gd-NBCB-TTDA-Legumain, to image the activity of asparaginyl
endopeptidase, known as Legumain, in a tumor, noninvasively using MR
imaging. The T1 relaxivity of Gd-NBCB-TTDA-Legumain was 4.94
mM-1 s-1 at 37± 0.1 °C and 0.47 T. Relaxometric studies shows that the
percentage change in T1 value of Gd-NBCB-TTDA-Legumain under the
different incubation time, increases 20 % in the presence of
3T3-Legumain cell lysate. The cytotoxicity results displayed that the cell
viability was more than 85% after the incubation of cells for 24 h under
high concentration of Gd-NBCB-TTDA-Legumain. The contrast agent
would be specifically recognized by Legumain enzyme around the tumor tissue, 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 murine colon carcinoma xenografts for MR images. Therefore, the
Gd-NBCB-TTDA-Legumain can be potentially used as a contrast agent
for cancer molecular imaging.

摘要 ............................................................................................................. I
Abstract ..................................................................................................... II
誌謝 ........................................................................................................... III
目錄 .......................................................................................................... IV
圖目錄 ...................................................................................................... IX
表目錄 .................................................................................................... XIII
一、前言 ..................................................................................................... 1
1.1 磁振造影(MRI) ........................................................................ 1
1.2 磁振造影成像原理 ................................................................... 2
1.3 磁振造影對比劑 ....................................................................... 3
1.4 磁振造影對比劑之發展簡史 ................................................... 3
1.5 磁振造影對比劑之種類 ........................................................... 4
1.5.1 順磁性磁振造影對比劑 ................................................. 4
1.5.2 超順磁性磁振造影對比劑 ............................................. 4
1.6 順磁性磁振造影對比劑之種類及研究現況 ........................... 5
1.6.1 離子性對比劑 ................................................................. 5
1.6.2 非離子性對比劑 ............................................................. 6
1.6.3 肝膽造影對比劑 ............................................................. 7
1.6.4 血池造影對比劑 ............................................................. 9
1.6.5 具生物活性之對比劑 ................................................... 11
1.6.6 目標化對比劑 ............................................................... 13
1.7 研究目的與動機......................................................................... 18
二、 儀器與試藥 .................................................................................... 20
2.1 儀器 (Instrumentation) ............................................................ 20
2.2 試藥 (reagents) .......................................................................... 22
三、實驗方法 .......................................................................................... 24
3.1 製備NBCB-NCS-5est (7) ......................................................... 25
3.1.1 C-(1-aminomethyl-cyclobutyl)-methylamine (CB-amine,
1)之製備[30] ................................................................................. 25
3.1.2 p-nitrophenylalanine methylester (NBPDA, 2)之製備[30]
..................................................................................................... 26
3.1.3 2-amino-N-(1-aminomethyl-cyclobutyl)-3-(4-nitrophenyl)-
propionamide (NBCB, 3)之製備[30].......................................... 26
3.1.4 NBCB-NO2-5est (5)之製備 .............................................. 27
3.1.5 NBCB-NH2-5est (6)之製備 .............................................. 28
3.1.6 NBCB-COOH-5est (7) 之製備 ....................................... 29
3.2 製備胜肽合成所需之溶劑 ......................................................... 30
3.2.1 偶合劑之製備 .................................................................... 30
3.2.2 活化劑之製備 .................................................................... 30
3.2.3 封端劑(capping agent)之製備.......................................... 30
3.2.4 Kaiser test 所需之溶劑製備 ............................................ 31
3.2.5 切除試劑及去保護基之溶劑製備 ................................... 31
3.2.6 胜肽序列的合成 ................................................................ 31
3.3 Legumain 胜肽之合成 ............................................................... 34
3.4 NBCB-TTDA-Legumain 之合成 .............................................. 36
3.4.1 NBCB-TTDA-Legumain 的合成 .................................... 37
3.4.2 切除resin 與保護基 ........................................................ 37
3.4.3 NBCB-TTDA-Legumain 純化與鑑定 ............................ 38
3.5 NBCB-TTDA-Legumain 釓及銪金屬錯合物之合成 .............. 38
3.6 內層水分子數之測定 ................................................................ 39
3.7 弛緩率(relaxivity)的測定 .......................................................... 39
3.8 Recombinant Human Legumain(rhLegumain)酵素之活化 .. 39
3.9 酵素誘導切割之弛緩時間(T1)測定 .......................................... 40
3.10 細胞株及其培養........................................................................ 41
3.11 Gd-NBCB-TTDA-Legumain 細胞毒性實驗 .......................... 41
3.12 Gd-NBCB-TTDA-Legumain 體外(in-vitro) MR 影像實驗 .. 41
3.13 Gd-NBCB-TTDA-Legumain 體內(in-vivo) MR 影像實驗 ... 42
四、結果與討論 ...................................................................................... 43
4.1 NBCB-COOH-5est 合成反應探討 ............................................ 43
4.2 胜肽合成機制之探討 ................................................................. 44
4.2.1 Resin 的去保護 ................................................................. 44
4.2.2 胺基酸的活化 ................................................................... 45
4.2.3 胺基酸與resin 的偶合 .................................................... 45
4.2.4 Capping ............................................................................. 46
4.2.5 反應完的resin 保存 ........................................................ 46
4.2.6 Kaiser test ......................................................................... 47
4.3 切割型磁振造影對比劑Gd-NBCB-TTDA-Legumain 之探討
............................................................................................................ 48
4.3.1 Legumain peptide 的鑑定 ................................................ 48
4.3.2 製備NBCB-TTDA-Legumain 合成反應機制 .............. 50
4.3.3 NBCB-TTDA-Legumain 純化及鑑定 ............................ 51
4.3.4 Gd-NBCB-TTDA-Legumain 純化及鑑定 ........................ 52
4.4 內層水分子數之探討 ................................................................ 54
4.5 釓錯合物之弛緩率之研究 ........................................................ 55
4.6 酵素誘導切割之弛緩時間(T1)測定 .......................................... 56
4.7 Gd-NBCB-TTDA-Legumain 之細胞毒性研究 ....................... 59
4.8 Gd-NBCB-TTDA-Legumain 體外(in-vitro) MR 影像實驗 .... 61
4.9 Gd-NBCB-TTDA-Legumain 體內 (in-vivo) MR 影像實驗 ... 63
五、結論 ................................................................................................... 64
六、參考文獻 .......................................................................................... 65
附錄 ........................................................................................................... 68

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