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。

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06454300/
• 关键词: THREE-DIMENSIONAL CULTURE; NEONATAL HEPATOCYTE; CHOLESTASIS; 培養肝細胞; 新生仔; 新生児; スフェロイド肝細胞; 毛細胆管; 母乳; 上皮増殖因子; 肝内胆汁うっ滞; エストラジオール; EGF

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，对肝内胆汁淤积的机制进行了研究，以阐明未成熟肝脏肝脏特异性功能的发育变化。在本研究中，我们建立了一种类似于体内三维结构的新生儿肝细胞球形培养模型，并在该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。培养14天后，球形培养肝细胞的白蛋白分泌量高于普通培养肝细胞。在胶原胶夹心培养系统中培养的肝细胞也保持了肝细胞的立方体形状和高水平的白蛋白分泌，这种培养体系与体内一样具有三维结构。用罗丹明-鬼笔环素进行肌动蛋白染色，可观察到相邻肝细胞之间的胆小管纤毛结构。胆汁淤积剂导致这些小管扩张，从而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，这些培养体系将有助于研究新生儿期肝脏胆汁淤积的机制。

项目成果

Fukunaga M: "The effects of inflammatory cytokines on the acute-phase response and DNA synthesis of neonatal Rat hepatocytes in primary culture." J Yonago Med Ass. 47. 103-115 (1996)

Fukunaga M：“炎症细胞因子对原代培养的新生大鼠肝细胞急性期反应和 DNA 合成的影响。”

Kono Y.: "Effects of human neonatal serum on DNA synthesis in suckling and adult rat hepatocytes in primary culture." Acta Paediatri Jpn. 36・1. 465-471 (1994)

Kono Y.：“人新生儿血清对原代培养的乳鼠和成年大鼠肝细胞的影响”Acta Paediatri Jpn 36・1（1994）。

Kono Y.: "Effects of cholestatic agents on the structure and function of bile canaliculi in neonatal rat hepatocytes in primary culture." Tohoku J Exp Med. 181・1. 9-18 (1997)

Kono Y.：“胆汁淤积剂对原代培养的新生大鼠肝细胞的结构和功能的影响”，Tohoku J Exp Med 181・1（1997）。

Kono Y,Fukunaga M,Shiraki K,Mura T: "Effects of human neonatal serum on DNA synthesis in suckling and adult rat hepatocytes in primary culture." Acta Paediatri Jpn. 36. 465-471 (1994)

Kono Y、Fukunaga M、Shiraki K、Mura T：“人新生儿血清对原代培养中的乳鼠和成年大鼠肝细胞 DNA 合成的影响。”

Yumi KOHNO,Maki FUKUNAGA,Kazuo SHIRAKI,Tetsuo MURA: "Effects of human neonatal serum on DNA synthesis in Suckling and adult rat hepatocytes in primary culture" Acta Paediatrica Japonica. 36. 465-471 (1994)

Yumi KOHNO、Maki FUKUNAGA、Kazuo SHIRAKI、Tetsuo MURA：“人新生儿血清对原代培养中乳鼠和成年大鼠肝细胞 DNA 合成的影响”Acta Paediatrica Japonica。