标题: 開發「活體動物自體螢光成像技術」應用於大鼠肝臟缺血再灌流傷害研究
Probing Hepatic Ischemia-Reperfusion of Rats with Intravital Autofluorescence Microscopy
作者: 呂學翰
Lu, Hsueh-Han
廖奕翰
Liau, Ian
應用化學系分子科學碩博士班
关键字: 缺血;再灌流;自體螢光;黃素;活體螢光顯微術;肝臟;小鼠肝細胞(FL83B);粒線體;Ischemia;Reperfusion;Autofluorescence;Flavin;Intravital autofluorescence microscopy;Liver;Mouse hepatocytes (FL83B);mitochondria
公开日期: 2010
摘要: 缺血再灌流損傷 (ischemia-reperfusion injury) 是指暫時缺血的器官組織在恢復血液供給後加劇傷害的現象。缺血再灌流損傷是腦中風、心肌梗塞或是器官移植手術的主要併發症。利用光學顯微鏡檢查切片的病理組織是目前判斷缺血再灌流損傷的主要方法。病理切片檢查只能提供損傷後組織傷害的嚴重程度,但無法提供即時資訊幫助醫師或研究人員判斷缺血的器官在再灌流後不同階段的損傷情形;此外,執行切片的過程也無可避免會造成組織額外的損傷。在此,我們改裝雷射掃描式共軛焦顯微鏡 (confocal laser scanning microscope),開發「活體動物光學成像系統」,以大鼠為模型動物,研究肝臟的缺血再灌流傷害。我們應用波長445 nm之雷射作為光源,激發並記錄肝臟組織內細胞之自體螢光訊號以形成影像,即時觀察大鼠肝臟在缺血再灌流過程之變化。我們的結果顯示,肝臟組織的整體螢光強度在缺血階段會快速的下降至基線值 (base line) 的50 % ∼ 60 %,而在恢復血液供給的階段,整體螢光強度會逐漸上升。螢光強度回復的速度以及程度與缺血時間長短相關。短暫缺血 (20分鐘) 時,螢光強度約在10分鐘内逐漸上升至基線值的90 %,組織也不會出現微血管堵塞的無復流 (no-reflow) 現象;而組織長時間缺血 (> 60 分鐘) 時,螢光強度的上升較慢,上升的趨勢也較不規律。更重要的是在長時間缺血後,我們直接觀察到部份組織出現間歇性甚至長時間的微血管堵塞。為釐清自體螢光的來源以及螢光在缺血再灌流傷害過程變化的原因,我們也建立細胞的灌流系統在體外 (in vitro) 模擬缺血再灌流過程,觀察肝細胞株在缺氧再灌流條件下之自體螢光變化。我們發現在添加黃素蛋白 (flavoprotein) 抑制劑 (diphenyleneiodonium chloride) 與粒線體電子傳遞鏈抑制劑 (antimycin A) 後,自體螢光約有40 %的下降。此結果說明,在我們的實驗條件之下,粒線體中的黃素蛋白是細胞自體螢光的主要來源。比較肝臟組織、肝細胞以及黃素分子 (flavin) 的螢光光譜也支持此假說。根據以上結果我們更進一步假設動物實驗觀察到的自體螢光變化是由於粒線體中黃素蛋白的黃素分子在缺血再灌流過程,在可發螢光的氧化態與不可發螢光的還原態之間轉換所造成。
Ischemia-reperfusion injury refers to the increased damage occurring to the tissue during reperfusion after a period of ischemia. Ischemia-reperfusion has long been recognized as the major complication of cardiac arrest, stroke, and organ transplantation, and remains one of the most active topics in fundamental biomedical research. The ability to evaluate timely the damage of tissue caused by ischemia-reperfusion is essential to understand to the pathogenesis. The knowledge produced is also important for the development of interventions to prevent or cure ischemia-reperfusion injury. By far, post-reperfusion histopathological examination remains a commonly employed method to assess ischemia-reperfusion injury. We have developed a minimally invasive, label-free, and real-time means to assess ischemia-reperfusion injury. By using an intravital microscope, we have obtained time-lapse autofluorescence images of rat liver during ischemia and reperfusion. We found the autofluorescence of the liver tissue decreased by 40-50 % rapidly during ischemia, but restored gradually during reperfusion. The rate and extent of recovery of the autofluorescence intensity during reperfusion depended strongly on the duration of ischemia. The autofluorescence intensity recovered to 90 % of the base line asymptotically and rapidly (<10 min) for short ischemia (20 min). In contrast, the recovery of the autofluorescence images exhibited heterogeneous patterns and the rate of recovery was significantly slower for long ischemic time (> 60 min). Moreover, we observed occasional or long-lasting no-reflow in some regions of this tissue. To elucidate the origin of the autofluorescence and to account the observation, we employed control experiments with inhibitors of mitochondria and flavin, and the results strongly suggested that the autofluorescence was produced from flavin bound to mitochondrial proteins. Based on the results, we further hypothesized that the spatiotemporal variation of autofluorescence images observed during ischemic-reperfusion was attributed to the conversion of flavin between the non-fluorescent reduced state and fluorescent oxidized state.
URI: http://140.113.39.130/cdrfb3/record/nctu/#GT079758516
http://hdl.handle.net/11536/46106
显示于类别:Thesis


文件中的档案:

  1. 851601.pdf

If it is a zip file, please download the file and unzip it, then open index.html in a browser to view the full text content.