| 研究課題/領域番号 |
22K20643
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| 研究種目 |
研究活動スタート支援
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| 配分区分 | 基金 |
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| 審査区分 |
0701:分子レベルから細胞レベルの生物学およびその関連分野
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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 – 2025-03-31
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| 研究課題ステータス |
交付 (2023年度)
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| 配分額 *注記 |
2,860千円 (直接経費: 2,200千円、間接経費: 660千円)
2023年度: 1,430千円 (直接経費: 1,100千円、間接経費: 330千円)
2022年度: 1,430千円 (直接経費: 1,100千円、間接経費: 330千円)
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| キーワード | exosomes / nanobody phage library / ischemic heart disease / targeted delivery / phage screening / biopanning / hypoxia / cell engineering / ischemic heart targeted / mesenchymal stem cells / Extracellular Vesicles / Mesenchymal Stem Cells / Myocardial Infarction / Regeneration / Nanobody Phage Library |
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| 研究開始時の研究の概要 |
1) Identify targeting nanobodies by in vivo biopanning of phage display nanobody library in MI mice
2) Engineer MSC-derived EVs with targeting nanobodies
3) Evaluate the therapeutic effects of modified EVs for the treatment of myocardial infarction
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| 研究実績の概要 |
Cardiomyocytes were treated with 1% O2, 5% CO2, 94% N2 for 48 hours. Membrane proteins of normoxic and hypoxic cardiomyocytes were isolated by Mem-PER Plus Membrane Protein Extraction Kit and then visualized on stain-free gel. Differential bands on stain-free gel were cut out and proceeded with Mass Spectrometry analysis. Totally 449 proteins were identified, and differential proteins were sorted out by fold change (>1.5) and P value (<0.05). Combined with protein functions under hypoxia, CD29 and CD166 were selected as candidate proteins for the further experiments. CD29 and CD166 were first biotinylated and then biotinylated CD29 and CD166 were immobilized on Maxisorb plate. After 2 rounds of target binding, phage binding, washing, elution and amplification, enriched phages were collected and processed for Next Generation Sequencing.
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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
We are working on the bioinformatic analysis of Next Generation Sequencing data.
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| 今後の研究の推進方策 |
Next Generation Sequencing will be analyzed by bioinformatics. Peptide sequences with high affinity and binding capacity to CD29 or CD166 will be selected and testified by in vitro and in vivo experiments.
Targeting peptide sequence will be inserted into an enriched protein, lysosome-associated membrane protein-2b (LAMP2b), on EV surface by genetic modification. I will investigate the biodistribution of targeting peptide-LAMP2b-EVs 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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