利用非侵入式多光子顯微術觀察脂多醣類(LPS)誘發老鼠之肝臟發炎

Abstract

台灣有四百萬人為B型肝炎的帶原者，而慢性肝炎甚至肝癌都有可能導致致命的傷害，根據行政院衛生署統計，癌症為十大死因之首，而肝癌又排名在男性癌症死因的第一名，慢性B肝或慢性C肝亦是導致肝癌或其他肝臟病變的重要原因，遺憾的是肝臟的問題在早期階段患者往往感受不到任何的病痛而延誤就醫的時間，因此尋求一個快速有效且對患者產生最低疼痛的診斷方式，及一種觀察肝臟活體代謝的研究模式，多光子顯微術也許將成為最佳的選擇。另外，傳統上為獲得生物組織的生理學資料，往往選擇有高解析度影像的切片技術，但其過程須以化學方式處理固定卻導致無法取得活體內的即時影像，且僅能獲得二維的資料，而本實驗室發展之肝臟視窗平台成為上述諸多限制的一大突破，本研究即以雙光子顯微術結合肝臟視窗平台，即以非侵入式方式觀察內毒素(脂多醣類-LPS)誘發老鼠肝臟發炎的即時反應。在取得的影像中我們觀察到Rhodamine-6G染色之白血球在免疫系統反應中首先聚集於肝臟，並開始滾動及附著於血管壁，接著穿透血管及細胞壁進入發炎組織，我們也發現不僅發炎後不同的肝細胞6-CFDA代謝速率之不同，即使未發炎老鼠的不同肝細胞亦有不同代謝速率。我們更期望我們的發展技術對未來肝臟甚至其他活體器官的觀察研究有所啟發及貢獻。

The parenchyma of liver has a highly organized and densely crowded sinusoidal system. They are tiny capillary-like vessels, therefore leukocytes and other blood cells must traverse through these vessels in contact with the endothelial cells flanking the sinusoids. By the use of intravital two-photon microscopy and hepatic imaging chamber we can study the response in vivo after administration of lipopolysaccharide (LPS), which is commonly used to model inflammatory mechanism. Whereas it is a critical and apparent process of the inflammatory response that the leukocytes recruitment at the inflamed tissues, these leukocytes roll along the vascular wall until they encounter stimulus produced by the endothelium cells and macrophages. The fluorescence micrographs of the hepatic microcirculation show that leukocytes accumulate within the sinusoids, then transmigrate across endothelial layer and into the inflammatory hepatocytes. We can also observe the metabolism of 6-CFDA within hepatocytes and the subsequent excretion in bile canaliculi.