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

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。在这项研究中，我们建立了一个类似于体内三维结构的新生儿肝细胞球形培养模型，并在模型中检测了肝细胞的功能，如蛋白质分泌和胆管的形成。新生儿肝细胞在涂有聚甲基丙烯酸羟乙酯的培养皿中培养5天后形成半球状体。它们在涂有BSA的盘子上形成完整的球体。培养14天后，球形培养肝细胞的白蛋白分泌水平高于普通培养肝细胞。在具有活体三维结构的拼贴凝胶夹层培养体系中培养的肝细胞，在14天内也保持了肝细胞的立方体形状和高水平的白蛋白分泌。罗丹明-phalloidin肌动蛋白染色观察相邻肝细胞间胆管样结构。抑胆剂引起这些小管扩张。因此，建立了新生儿肝细胞的三维培养。由于肝细胞维持较高的肝脏特异性功能和形态，这些培养系统应该有助于研究新生儿期肝脏胆汁淤积的机制。

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.