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Development of bio-artificial liver by the mass transfer in the small hepatocytes

通过小肝细胞内的传质开发生物人工肝

基本信息

批准号：
12555060
负责人：
TANISHITA Kazuo
金额：
$ 8.64万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

The morphogenesis and movement of bile canaliculi (BC) are not well understood. This is because culture of hepatocytes that maintain polarity of cell membranes and possess highly differentiated functions has never been successful. We found that small hepatocytes (SHs), which are known to be hepatic progenitor cells, could proliferate and differentiate into mature hepatocytes and that BC-like structures developed between rising/piled-up cells. In the present experiment we examined how SHs could form BC-like structures in the process of their maturation, whether the structures could be functionally active as BC, how BC-like structures contract to expel substances, and whether A23187 and Endothelin-1 caused BC contractions or not.The results of immunocytochemistry, immunoblots, and immunoelectron micrographs revealed that BC proteins localized in the intercellular space that coincided with BC-like structures formed between rising/piled-up cells. Fluorescein was secreted into BC and accumulated without leakage. Time-lapse microscopy showed that continuous contraction and dilatation occurred in the BC-like structures, and fluorescent dye was secreted into the reformed BC and unidirectionally carried through the structures. We also found out BC contractions occurred in a coordinated manner, which at least a few cells length of the BC-like structures contract synchronously. It is also found out that both A23187 and Endothelin-1 caused BC contractions. The functional abilities of reformed BC may be equivalent to those of in vivo BC.

胆管（BC）的形态发生和运动尚不清楚。这是因为维持细胞膜极性并具有高度分化功能的肝细胞的培养从未成功过。我们发现小肝细胞（SHs），已知是肝祖细胞，可以增殖并分化为成熟肝细胞，并且在上升/堆积的细胞之间形成bc样结构。在本实验中，我们研究了SHs在成熟过程中如何形成BC样结构，这些结构是否具有BC的功能活性，BC样结构如何收缩以排出物质，以及A23187和内皮素-1是否引起BC收缩。免疫细胞化学、免疫印迹和免疫电镜结果显示，BC蛋白定位于细胞间隙，与上升/堆积细胞之间形成的BC样结构相吻合。荧光素分泌到BC中，积累无渗漏。延时显微镜显示，BC样结构发生持续收缩和扩张，荧光染料分泌到改造后的BC中，并单向通过结构携带。我们还发现BC的收缩是以协调的方式发生的，至少有几个细胞长度的BC样结构同步收缩。同时发现A23187和内皮素-1均引起BC收缩。重组BC的功能能力可能与体内BC相当。