A study on mechanisms of intrahepatic cholestasis using neonatal rat hepatocytes cultured in three-dimensions.

利用三维培养新生大鼠肝细胞研究肝内胆汁淤积机制。

基本信息

批准号：
06454300
负责人：
SHIRAKI Kazuo
金额：
$ 3.65万
依托单位：
Tottori University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

项目摘要

We have investigated on the mechanisms of intrahepatic cholestasis to elucidate developmental changes of liver-specific functions in the immature liver using neonatal rat hepatocytes in primary culture. In this study, we developed a spheroidal culture model of neonatal hepatocytes which resembles three-dimensional structure in vivo and examined hepatocellular functions such as protein secretion and formation of bile canaliculi in the model.Neonatal hepatocytes formed semispheroids after 5 days cultured on dishes coated with polyhydroxyethyl methacrylate. They formed complete spheroids on the dishes coated with BSA.Spheroidal cultured hepatocytes maintained higher levels of albumin secretion than plain cultured hepatocytes after 14 days in culture. Hepatocytes cultured in collage gel sandwich culture system, which provides three-dimensional structures as in vivo, also maintained cuboidal shape of hepatocytes and high levels albumin secretion for 14 days. Bile canaliculilike structure was observed between adjacent hepatocytes by actin staining using rhodamine-phalloidin. Cholestatic agents caused dilatation of these canaliculi.Thus, three-dimensional culture of neonatal hepatocytes was established. As hepatocytes maintain higher of liver-specific function and morphology, these culture systems should be useful for studying the mechanisms of hepatic cholestasis in the neonatal period.

我们利用原代培养的新生大鼠肝细胞研究了肝内胆汁淤积的机制，以阐明未成熟肝脏中肝脏特异性功能的发育变化。本研究建立了类似于体内三维结构的新生儿肝细胞球状培养模型，并在模型中检测了肝细胞的蛋白质分泌和胆小管形成等肝细胞功能。新生儿肝细胞在涂有蛋白质的培养皿上培养5天后形成半球状。聚甲基丙烯酸羟乙酯。它们在涂有BSA的培养皿上形成完整的球体。培养14天后，球状培养的肝细胞比普通培养的肝细胞保持更高水平的白蛋白分泌。在胶原凝胶夹心培养系统中培养的肝细胞，提供了与体内一样的三维结构，也保持了肝细胞的立方体形状和高水平的白蛋白分泌14天。通过使用罗丹明-鬼笔环肽进行肌动蛋白染色，在相邻肝细胞之间观察到胆小管样结构。胆汁淤积剂导致这些小管扩张。由此建立了新生儿肝细胞的三维培养。由于肝细胞保持较高的肝脏特异性功能和形态，这些培养系统应可用于研究新生儿期肝胆汁淤积的机制。