| 研究課題/領域番号 |
22K20643
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| 研究機関 | 医療法人徳洲会湘南鎌倉総合病院(臨床研究センター) |
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研究代表者 |
ヤン ジュンジー 医療法人徳洲会湘南鎌倉総合病院(臨床研究センター), 湘南先端医学研究所 再生医療開発研究部, 主任研究員 (20962137)
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| 研究期間 (年度) |
2022-08-31 – 2024-03-31
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| キーワード | nanobody phage library / biopanning / hypoxia / cell engineering / ischemic heart targeted / mesenchymal stem cells / exosomes |
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| 研究実績の概要 |
We have generated a novel modular synthetic nanobody phage display library to select for targets including CD8, PD-1 and HER2. We also identified differential proteins of normoxic and hypoxic cardiomyocytes by liquid chromatography mass spectrometry. Among these differential proteins, CD29 and CD166 were identified to have most significant difference and also be involved in cell survival in hypoxic conditions. CD29 and CD166 will be chosen for further experiments.
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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
CD29 and CD166 are biotinylated and immobilized on Maxisorb plate. Targeting binding, followed by phage binding, washing, elution and amplification are being performed to enrich phage library on CD29 or CD166. Next-generation sequencing will be performed to select most enriched phages.
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| 今後の研究の推進方策 |
Engineer mesenchymal stem cell-derived exosomes with targeting nanobodies and evaluate the therapeutic effects of modified exosomes for the treatment of myocardial infarction. Targeting nanobody will be inserted into an enriched protein, lysosome-associated membrane protein-2b (LAMP2b), on exosome surface by genetic modification. Through forced overexpression of targeting nanobody-LAMP2b in mesenchymal stem cells (MSCs), exosomes secreted by the cells will have targeting nanobody-LAMP2b on their surface. Targeting nanobody-LAMP2b-exosomes will be investigated in vivo by live imaging and multimodal imaging and evaluate the mechanisms and effectiveness for treating MI in mouse models through assessments of cell survival, neovascularization, infarct size, inflammation, and cardiac function.
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| 次年度使用額が生じた理由 |
In the previous fiscal year, the collaborator conducted generation and validation of nanobody phage display library, and identification of differential proteins of normoxic and hypoxic cardiomyocytes by liquid chromatography mass spectrometry. In this fiscal year, we will engineer mesenchymal stem cell-derived exosomes with targeting nanobodies and evaluate the therapeutic effects of modified exosomes for the treatment of myocardial infarction in a mouse model.
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