肝移植におけるsiRNAと機械灌流法を用いたシグナル遮断による虚血再灌流傷害抑制

肝移植中使用siRNA和机器灌注信号阻断抑制缺血再灌注损伤

• 批准号: 22K16436
• 负责人: 藤好 真人
• 金额: $ 3万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Early-Career Scientists
• 财政年份: 2022
• 资助国家: 日本
• 起止时间: 2022-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-22K16436/
• 关键词: 肝移植; 機械灌流; 核酸治療; 虚血再灌流傷害; 臓器保存

当研究では体外機械灌流法を導入したマウス肝移植モデルを実験系として用いる。前実験において、リポフェクション法を用いた肝臓へのsiRNAトランスフェクションは数時間で行うことができる一方で非生理的低温下では導入効率が著しく低下することが分かっている。そのため、本研究においてsiRNAの体外導入は37℃での常温機械灌流を介して行う予定である。マウス肝移植において常温機械灌流を導入した報告はこれまでにないため、当研究ではまずその技術的確立が必要となる。我々はこれまでに低温機械灌流およびsub-normothermic machine perfusionには成功しているが、これらに比べて常温機械灌流は酸素担体による酸素送達、灌流液の品質維持、灌流ダイナミクスの精密な制御など技術的要求が高く、今年度はその条件検討に多くの時間を費やした。常温機械灌流では低温機械灌流に比べ、より正確な門脈灌流圧測定とポンプコントロールを要するため、灌流装置の改良を行った。酸素担体として赤血球を含有する灌流液を充分量作成することはマウスでは難しいため、血液ドナーからの高効率の赤血球採取法の開発および灌流回路の小ボリューム化を進めた。灌流温度の上昇と灌流液の小ボリューム化は灌流中における灌流液の急激な品質低下につながるため、その条件検討に多くの時間を費やしている。灌流回路については、横隔膜を合併摘出し、肝上部下大静脈を閉鎖する術式によりドレナージ系を閉鎖回路化することにより灌流液ボリュームの大幅な減量を可能とした。

When in vitro mechanical perfusion was introduced into the liver transplantation system, In the past, the siRNA was introduced into the liver at a low temperature after several days of operation. In this study, siRNA was introduced into the cells at 37℃ by mechanical perfusion. The introduction of normothermic mechanical perfusion into liver transplantation is reported, and it is necessary to establish the technique when studying it. In response to this, the requirements of cryogenic mechanical perfusion and sub-normothermic machine perfusion are higher than those of normal temperature mechanical perfusion, such as acid delivery, quality maintenance of perfusate, precision control of perfusion, and more time for discussion of this year's conditions. Mechanical Perfusion at Normal Temperature Compared with Mechanical Perfusion at Low Temperature, Accurate Measurement of Portal Perfusion Pressure and Improvement of Perfusion Device The preparation of sufficient amount of acid carrier and erythrocyte in perfusion fluid, the development of erythrocyte collection method with high efficiency and the improvement of perfusion circuit. The rise of perfusion temperature and the decrease of perfusion fluid quality during perfusion, the decrease of perfusion fluid quality during perfusion, the increase of perfusion fluid temperature and the decrease of perfusion fluid quality during perfusion, the increase of perfusion fluid temperature during perfusion, the decrease of perfusion fluid quality during perfusion, the decrease of perfusion fluid temperature during perfusion, the decrease of perfusion fluid quality during perfusion, the decrease of perfusion fluid temperature during perfusion, the decrease of perfusion fluid temperature during perfusion, the decrease of perfusion fluid quality during perfusion, the decrease of perfusion fluid temperature during perfusion, the decrease of perfusion fluid temperature during perfusion, the decrease of perfusion fluid quality during perfusion, the decrease of perfusion fluid temperature during perfusion, the decrease of perfusion fluid temperature during perfusion, the decrease of perfusion fluid temperature during perfusion, the decrease of perfusion fluid temperature during perfusion, the decrease of perfusion fluid quality during perfusion, the decrease of perfusion fluid temperature during perfusion, the decrease of perfusion, the decrease of perfusion fluid temperature during perfusion, the decrease of perfusion, the decrease of perfusion fluid temperature during perfusion, the decrease of perfusion, the decrease of perfusion fluid temperature during perfusion, the decrease of perfusion, the decrease of perfusion fluid quality during perfusion, and the increase of perfusion temperature during perfusion. Perfusion loop closure, diaphragm closure, closure of superior hepatic vein closure, closure of perfusion loop closure, and significant reduction in perfusion volume.