| 研究課題/領域番号 |
16F16109
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| 研究機関 | 東京大学 |
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研究代表者 |
浦野 泰照 東京大学, 大学院薬学系研究科(薬学部), 教授 (20292956)
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| 研究分担者 |
RIVAS CHARLOTTE 東京大学, 薬学研究科(研究院), 外国人特別研究員
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| 研究期間 (年度) |
2016-10-07 – 2019-03-31
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| キーワード | Sialidase / Fluorescence activation / Lung cancer / Self-immolative space |
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| 研究実績の概要 |
A novel fluorescence probe that is able to report on the presence of sialidase has been synthesised and evaluated as a cancer detection tool. The probe, HMRef-S-Neu5Ac is an improvement on sialidase probes previously developed in this project as it has greater stability in solution, in particular to hydrolysis via an acid-mediated pathway (i.e. in the absence of the target enzyme sialidase). This was achieved by incorporating a self-immolative spacer group between the sialic acid residue and the HMRef fluorophore. Kinetic measurements have shown the probe to have similar properties to that of a commercially available sialidase substrate, 4-MUNANA, demonstrating that the spacer group does not impede substrate recognition. The probe has been assessed in prostate cancer cells (LNCaP, DU145, PC3) which are reported to have significant levels of sialidase. Fluorescence activation was observed, with localisation being mainly in the cell membrane. The probe has also shown to be able to distinguish between normal and tumour types of human lung cancer specimens and so further experiments are ongoing to investigate sialidase levels in this cancer type.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
A patent is being prepared detailing the synthesis, properties and biological results of HMRef-S-Neu5Ac. Further biological experiments are currently being carried out to add to this patent. These include qPCR experiments to confirm the expression levels of NEU1-4 in the clinical lung cancer samples. If a difference in expression is observed between cancerous and normal samples, validation experiments using antibodies and positive controls, as well as Western Blot experiments will be carried out. If no significant expression is observed in the tumour samples, a DEG assay will be employed to determine what enzymes may be activating the probe (assay which utilises the combination of native polyacrylamide gel electrophoresis with a multiwell-plate-based fluorometric assay to find protein spots with the specified activity).
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| 今後の研究の推進方策 |
A derivative of HMRef-S-Neu5Ac will be synthesised as a probe to detect the influenza virus in clinical samples. Fluorescent substrates bearing a 4,7-dimethoxy derivative of sialic acid have been shown to have specificity for type A and B influenza and so a HMRef analogue is to be synthesised to give a probe which is capable of reporting on these influenza types using visible wavelength light. This would then be implemented in a digital detection kit for the influenza virus being developed in the Graduate School of Engineering in collaboration with the Noji lab. Current commercially available fluorescence probes being used for detection are unsuitable due to high background noise therefore a new probe based on the stable HMRef-S-Neu5Ac motif is required.
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