蛍光顕微鏡法によるマウス胆道樹の階層構造の精密可視化

Fine-scale visualizing the hierarchical structure of mouse biliary tree with fluorescence microscopy method.

• Yuwei Chen
• Lin Bai
• Yongjie Zhou
• Xiaoyun Zhang
• Jie Zhang
• Yujun Shi

抄録

肝臓は重要な臓器であり、肝小葉は肝細胞や胆道上皮細胞などの実質細胞が集まった最も基本的な構造的・機能的な単位である。胆道を構成する胆のう細胞の連続的な管状配列は肝機能にとって重要であるが、肝硬変や一部の先天性疾患など多くの肝疾患では胆道が障害されることが多い。胆道の微細・立体的な可視化は、これらの肝疾患の構造基盤の理解に役立つと考えられています。本研究では、食事給餌、手術、遺伝子組み換えにより、いくつかの胆道損傷マウスモデルを樹立した。肝実質細胞の細胞質と核は、それぞれ蛍光色素ローダミンB(赤)とHoechst(青)を積極的に取り込んで標識した。肝臓のエンブロック除去後、胆道を緑色蛍光色素であるフルオレセインイソチオシアネート（FITC）で逆流性に灌流した。その後、肝臓を共焦点顕微鏡下で観察した。胆道樹全体の微細構造および三次元（３Ｄ）構造、特に末期胆管のネットワークおよび隣接する肝細胞が明確に可視化された。胆道は、正常肝モデルと疾患肝モデルで明確な特徴を示した。以上のことから、我々はマウス肝臓に広がる胆道の微細で階層的な構造を可視化するためのシンプルで再現性の高いイメージング方法を開発した。

The liver is a vital organ and the hepatic lobule serves as the most basic structural and functional unit which is mainly assembled with parenchymal cells including hepatocytes and biliary epithelial cells. The continuous tubular arrangement of biliary cells which constitutes the biliary tracts is critical for liver function, however, the biliary tracts are often disrupted in many liver diseases such as cirrhosis and some congenital disorders. Visualization of the biliary tracts in fine-scale and three-dimension will help to understanding the structure basis of these liver diseases. In the present study, we established several biliary tract injury mouse models by diet feeding, surgery or genetic modification. The cytoplasm and nuclei of the parenchymal cells were marked by active uptake of fluorescent dyes Rhodamine B (red) and Hoechst (blue), respectively. After the removal of liver en bloc, the biliary tracts were retrogradely perfused with green fluorescent dye, fluorescein isothiocyanate (FITC). The liver was then observed under confocal microscopy. The fine-scale and three-dimensional (3D) structure of the whole biliary tree, particularly the network of the end-terminal bile canaliculi and neighboring hepatocytes were clearly visualized. The biliary tracts displayed clear distinct characteristics in normal liver and diseased liver models. Taken together, we have developed a simple and repeatable imaging method to visualize the fine-scale and hierarchical architecture of the biliary tracts spreading in the mouse liver.

© 2020 The Author(s).