應用活體多光子影像技術解析肝膽系統之生物物理現象

Applications of In Vivo Multiphoton Imaging In Elucidating Biophysical Phenomena of the Hepatobiliary System

李峰杰(Feng-Chieh Li)

指導教授 ： 董成淵

共同指導教授 ： 李宣書(Hsuan-Shu Lee)

國立臺灣大學/理學院/物理研究所/博士(2010年)

https://doi.org/10.6342/NTU.2010.01160

摘要

肝臟是人體中最大的臟器，像個化學工廠般的運作，其主司代謝、解毒、蛋白質及維生素合成及藥物轉化等功能。傳統上對於肝臟之生物物理、生理、及病理上的研究，主要仰賴組織切片技術較為靜態及間接，而活體上的直接性觀察較為缺乏，阻礙了活體中生理及病理之研究。有鑑於此，我們發展了一套活體觀測暨研究平台，除了動態影像之觀察外，尚包含了與肝臟健康息息相關的代謝及循環功能評估系統之建立。在動態的活體影像中，我們發現了肝細胞死亡過程中所經歷的過程，及關鍵的死亡契機，同時發展出肝細胞死亡的假說。對於肝臟之動態代謝功能的觀察，我們係以螢光染劑6-CFDA做為探測器，經由肝臟代謝之過程中，發現其螢光強度之時變曲線可經由簡單之數學模型做簡化及分析，進而發展出肝臟代謝模型，並於模型中獲得描述代謝能力之k1及k2等參數。我們利用了肝臟代謝模型於藥物型之肝臟急性衰竭之研究，並觀察其由受損至恢復過程k值之變化。活體動態影像中，經由血流出入之方向以判定肝小葉之分佈狀況，基於肝細胞內之運輸蛋白及酵素分佈差異進而影響6-CFDA的代謝速率，藉此可由代謝之k值分佈大略勾勒出acinus的外型。綜合目前我們所進行的工作，是利用多光子動態活體肝臟影像之研究，以定性及定量的方式分析肝膽微循環之變化具體肝臟受損狀況之關聯。

關鍵字

活體多光子顯微術 ； 肝膽代謝 ； 肝臟功能 ； 乙醯胺酚 ； 肝細胞死亡機制

並列摘要

Liver, the largest internal organ, is a major chemical factory in the body responsible for important functions such as metabolism, detoxification, nutrient storage, and serum protein production. Traditionally, the study of the liver depended heavily on histological techniques, which are limited to ex-vivo observations and lack dynamic information of the hepatobiliary system. In order to study hepatic metabolism in vivo, we have designed a hepatic imaging chamber made of biocompatible titanium alloy (6V4Al-Ti). Combining the chamber with multiphoton microscopy, we were able to monitor and quantitatively analyze the hepatobiliary metabolism to single cell resolution in vivo. In addition to studying metabolic dynamics, we applied our methodology to investigate mechanism of cell-death. We found that and determined a critical limit for hepatocyte death. We further developed a model based on first order rate equation to model the observed variation of 6-CFDA fluorescence intensity with time and estimate the metabolic capability of hepatocytes. Specifically, we found that zonal difference in the metabolic activity can be revealed by the distribution of the model parameters. Our approach allows the intravital observation of hepatic activities such as acetaminophen (APAP), common bile duct ligation (CBDL), Lipopolysaccharides (LPS), and CCl4. This thesis demonstrated that intravital multiphoton imaging could be used to reveal and quantify metabolic changes in the mouse liver and its potential application in studying many liver disorders.

並列關鍵字

intravital multiphoton microscopy ； hepatobiliary metabolism ； liver function ； acetaminophen ； mechanism of hepatocyte death

參考文獻

23 Dong, H. J., Haining, R. L., Thummel, K. E., Rettie, A. E. & Nelson, S. D. Involvement of human cytochrome P450 2D6 in the bioactivation of acetaminophen. Drug Metab Dispos 28, 1397-1400 (2000).

1 Denk, W., Strickler, J. & Webb, W. Two-photon laser scanning fluorescence microscopy. Science 248, 73 (1990).

3 Sandison, J. The transparent chamber of the rabbit's ear, giving a complete description of improved technic of construction and introduction, and general account of growth and behavior of living cells and tissues as seen with the microscope. American Journal of Anatomy 41, 447-473 (2005).

4 Liu, Y. et al. Visualization of hepatobiliary excretory function by intravital multiphoton microscopy. Journal of Biomedical Optics 12, -, doi:Artn 014014

Doi 10.1117/1.2710237 (2007).