應用先進光譜成像技術研究缺血再灌流傷害

Abstract

過去的動物實驗及臨床觀察發現恢復血液供給到缺血組織(例如腦中風、心肌梗塞或器官移植)會造成大量細胞壞死，或所謂「缺血再灌流傷害」。缺血再灌流傷害的致病機轉與在灌流初期產生之活性含氧物質 (reactive oxygen species) 氧化脂質或蛋白質等分子造成細胞氧化傷害，以及再灌流後部份微血管之「無復流 (no re-flow)」現象造成組織持續缺血等機制有關。過去幾年在國科會支持下，吾人在氧化傷害及相關基礎生醫研究已獲得以下初步成果: (1)建立觀測單一細胞發生脂質過氧化傷害之新方法 (J. Raman Spectro. 2009)；(2)應用光譜及影像技術分析巨噬細胞產生活性含氧物質破壞微生物之過程 (JACS 2010)；(3)剖析脂質分子氧化之結構變化與磷脂膜流動性改變之相關性 (JPC-B 2010)；(4)建構多模式多光子成像系統展現以無須染色方式分辨生物組織之成分 (Opt. Exp. 2008；PLoS One 2011)；(5)應用CARS 成像系統以非破壞方式定量肝臟組織中之脂肪含量 (Anal. Chem. 2009)；(6)開發感測活體細胞內活性含氧物質之新方法 (Anal. Chem. 2011)。 在以上基礎之下，吾人希望建立缺血再灌流傷害及相關疾病之實驗動物模型，並開發先進光學顯微鏡技術應用於疾病之基礎生醫研究。在第一年的計畫期間，吾人按照計畫的規化已完成或正在進行以下工作:(1)建立大鼠肝臟缺血再灌流的實驗動物模型(2)應用 mitochondrial flavoprotein 之自體螢光，觀測細胞 hypoxia-reoxygenation 過程之基礎工作(3)建立應用自體螢光動物動物顯微成像技術，觀測大鼠缺肝臟血再灌流過程(4)建立小型斑馬魚養殖系統(5)建立斑馬魚動脈粥狀硬化及腦中風之實驗動物模型

Ischemia-reperfusion injury refers to an increased tissue damage that occurs during reperfusion after a period of ischemia. The adverse effects of ischemia-reperfusion has been recognized as the major complication of cardiac arrest, stroke and also organ transplantation, and remains one of the most active research topics in fundamental and clinical research. The recognition of microvascular dysfunction and oxidative injury as two of the main pathogenic mechanisms of ischemia-reperfusion injury has led to extensive research effort aiming to reveal the interplay of these two factors with ischemia-reperfusion injury. Thanks to the generous support from the National Science Council, we have achieved the following specific objectives: (1) Application of Raman tweezers to assess cellular oxidative injury (J. Raman Spectro. 2009)； (2) Application of Raman microspectroscopy to assess the intraphagosomal oxidative damage of intruding microbe (JACS 2010)； (3) Examination of the alteration of membrane fluidity on membranes subject to oxidative attack (JPC-B 2010)； (4) Development of a multimodal multiphoton microscopy system (Opt. Exp. 2008； PLoS One 2011)； (5) Application of CARS microscopy to quantify the fat content of intact liver tissues (Anal. Chem. 2009)； (6) Development of a biosensor for the quantification of endogenous HOCl (Anal. Chem. 2011). In the first year of the three-year project, we have accomplished or are performing the following tasks: (1) To establish a rat model of ischemia and reperfusion (2) To apply the autofluorescence of mitochondrial flavoprotein to study hypoxia-reoxygenation of cells (3) To apply intravital autofluorescence microscopy to study hepatic ischemia and reperfusion on the rat model (4) To establish a small aqua system of zebrafish (5) To establish a zebrafish model of atherosclerosis and stroke