應用於Legumain偵測之含胜肽近紅外螢光分子
影像探針之研究

由於近紅外螢光比可見光能更有效率的穿透皮膚組織，因此已被運用至各種不同的生理研究中，更甚至將其運用於體內實驗。本研究欲開發與合成一條由8個胺基酸所組成的探針（probe），其針對 Legumain酵素具有目標性，且在多種腫瘤上都有過度表現。在胜肽兩端鍵結上Cy-TE777及Cy-TE807，以形成一個近紅外線螢光探針。這種近紅外線螢光探針在光學影像之體內（in vivo）實驗中，可作為酵素活性的偵測器。利用HPLC進行探針的純化後利用質譜儀鑑定其分子量為2440.92 g/mol，與實際分子量2441.17 g/mol相符。利用此胜肽受質上的Cy-TE807可吸收Cy-TE777的發射能量，達到抑制的效應 ( quench effect )，使得探針上的Cy-TE777放射能量（820 nm）於一開始有較低的螢光放射，最後我們將此探針與Legumain酵素反應之後，由螢光光譜儀觀察到Cy-TE777（820 nm）原本被抑制的螢光被釋放。在細胞毒性實驗中發現，在10μM Legumain probe培養下，細胞存活率仍達80%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。在體外（in vitro）實驗中可看出Legumain probe在加入有tranfection Legumain DNA的lysate後有明顯的訊號變亮，但在加入control lysate後也有少許的變亮，推測可能有少量非特異性之切割。在體內(in vivo)實驗研究中，Legumain probe可目標化至表現Legumain 之CT-26腫瘤且提高其訊號強度。結果顯示此光學影像探針有潛力對有Legumain表現之腫瘤進行診斷及追蹤。

關鍵字

近紅外線螢光探針；光學影像

並列摘要

Near-infrared fluorescence(NIRF) has been applied to image various biological events in vivo, because it penetrates tissue more efficiently than visible radiations. An 8-amino acids peptide substrate was designed and synthesized for specific enzyme Legumain. A near-infrared (NIR) cyanine fluorochrome serving as the optical imager can be used for in vivo imaging of enzyme activity. So we developed a probe that consisted of a target peptide ( legumain ) conjugated to an effector recognition sequence(AANL) that was flanked by a fluorophore-quencher pair (Cy-TE777 and Cy-TE807). HPLC was employed to purify the probe, and mass spectrometer was used to appraise the result. It’s molecular weight is 2440.92 g/mole, and intrinsical molecular weight is 2441.17 g/mole. Once exposed to Legumain enzyme, the recognition sequence was cleaved, resulting in separation of the fluorophore-quencher pair and signal generation. The probe was determined by measuring fluorescence emission at 840 nm. When the probe was cleaved by legumain enzyme, the Cy-TE777 emission 820nm was release. The cytotoxicity experiment showed cell incubated with 10μM Legumain probe, the cell viability can maintain up to 80%. In vitro study showed the probe incubated with Legumain enzyme, the signal intensity significant enhancement. But the probe incubated with control Legumain enzyme, the signal intensity also have a little arise. It seems to have some non-specific cleavage. The results of Legumain probe indicates that it has low cytotoxicity, and specific tumor localization. This optical probe has a potential for cancer molecular imaging with optical imaging.

並列關鍵字

Near-infrared fluorescence probe ； optical imaging

參考文獻

1.Liu, C.; Chengzao, S.; Huang H.; Kim J.; Thomas, E. Cancer Res. 2003, 63, 2957-2964.

Google Scholar

2.Maxwell, D.; Chang, Q.; Chang, Q.; Zhang X.; Edward, M. B.; David, P. W. Bioconjugate Chem. 2009, 20, 702-709.

Google Scholar

3.Bullok, K. E.; Maxwell, D.; Kesarwala, A. H.; Gammon, S.; Prior, J. L.; Snow, M.; Stanley,S.; and Piwnica-Worms, D. Bioconjugate Chem. 2007, 46, 4055-4065.