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A study on the Changes of localization of canalicular transporters in cholestasis.

胆汁淤积时小管转运蛋白定位变化的研究。

基本信息

批准号：
12670517
负责人：
TAKIKAWA Hajime
金额：
$ 1.98万
依托单位：
Teikyo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
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

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670517/
关键词：
bile acid organic anion canalicular transporter MRP2 BSEP cholestasis

项目摘要

Biliary excretion of bile acids and organic anions is mediated by ATP-dependent primary active transporters at the canalicular membrane. Multidrug resistance protein 2 (MRP2) is a transporter for organic anions such as conjugated bilirubin, and is defective in Dubin-Johnson syndrome. Bile salt export pump (BSEP) is a transporter for amidated bile acids, and is defective in progressive familial intrahepatic cholestasis type 2. Down regulation of MRP2 and BSEP has been reported in various cholestatic models. In addition, the impairment of vesicular targeting of transporters to the canalicular membrane has been postulated as an important mechanism of cholestasis. In the present study, in order to clarify the mechanism of cholestasis, localization ofMRP2 and BSEP in hepatocytes was investigated by immunohistochemistry in bile duct-ligated rats and lipopolysaccharide (LPS)-induced rat cholestasis. Polyclonal anti-MRP2 and anti-BSEP antibodies were obtained by the immunization of rabbits by C-terminal peptides coupled with KLH. Microscopic observation of the liver after the treatment with HRP-labeled second antibody revealed the localization ofMRP2 and BSEP at the canalicular membrane of hepatocytes. In bile duct-ligated rats, a decrease of the staining ofMRP2 and BSEP at the canalicular membrane was observed, suggesting a dysfunction of these transporters. In contrast, the staining of these transporters was the same as controls in LPS-induced cholestatic model. Similar results were obtained by using commercially available monoclonal anti-MRP2 antibody. In future, immunofluorescent observations by a confocal laser microscopy, which was insufficient in the present study, and studies on human liver with cholestasis are planed.

胆汁酸和有机阴离子的胆汁排泄由小管膜上的ATP依赖性初级主动转运蛋白介导。多药耐药蛋白2（MRP 2）是有机阴离子如结合胆红素的转运蛋白，在Dubin-Johnson综合征中有缺陷。胆盐输出泵（BSEP）是一种酰胺化胆汁酸的转运蛋白，在进行性家族性肝内胆汁淤积症2型中存在缺陷。在各种胆汁淤积模型中报告了MRP 2和BSEP的下调。此外，转运蛋白到小管膜的囊泡靶向受损已被假定为胆汁淤积的重要机制。本研究采用免疫组织化学方法，观察了胆管结扎大鼠和脂多糖（LPS）诱导的胆汁淤积大鼠肝细胞中MRP 2和BSEP的定位，以探讨胆汁淤积的发生机制。通过C端肽与KLH偶联免疫家兔，获得了抗MRP 2和抗BSEP的多克隆抗体。HRP标记的第二抗体处理后的肝脏显微镜观察显示，MRP 2和BSEP定位于肝细胞小管膜。在胆管结扎大鼠中，观察到小管膜上的MRP 2和BSEP染色减少，表明这些转运体功能障碍。相反，在LPS诱导的胆汁淤积模型中，这些转运蛋白的染色与对照相同。通过使用市售的单克隆抗MRP 2抗体获得了类似的结果。在未来，免疫荧光观察共聚焦激光显微镜，这是不够的，在目前的研究，并研究人类肝脏胆汁淤积的计划。