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Molecular biological assessment of hepatocyte damage and fibrosis in intractable pediatric liver diseases

难治性小儿肝病肝细胞损伤和纤维化的分子生物学评估

基本信息

批准号：
17591087
负责人：
MUSHIAKE Sotaro
金额：
$ 2.3万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591087/
关键词：
Grb10 Tyrosine kinase type receptor Hepatic fibrosis Hepatocyte damage Hepatic stellate cell SH2ドメイン肝硬変偽胆管

项目摘要

The Grb10 expression in human pathological cirrhotic liver tissues and the animal models with liver damages were assessed. Dimethylnitrosamine (DMN) administration and CCI4 administration mice models were constructed according to the former reported protocol but they were not utilized for the following experiments because of their high lethality and hepatocyte damage rather than portal fibrosis. Then immunohistochemical staining for Grb10 was performed on the common bileduct ligation (CBDL) model, but no significant staining was obtained. Examination of mRNA expression by RT-PCR in the liver tissue of the CBDL mouse and the normal adult mouse demonstrated coordinative expressions of Grb10. The result was different from the former report that the Grb10 expression in the adult mouse liver was very low as compared with that in the embryo. On the other hand, in Western blot, protein expression in the adult mouse liver was very low compared to those in fetus and CBDL mouse liver.Then hepatic parenchymal cells and hepatic stellate cells (HSC) were extracted from the rat liver, the mypfibroblastic transformation of HSC was assessed and the expressions of Grb10 were examined by RT-PCR. In result, the mRNA expressions were detected in myofibroblast (HSC), while very low in hepatocyte. In addition, mRNA expression was demonstrated in the damaged liver tissue of DMN-treated SD rats which is identical to the localization found in human cirrhotic liver. Though that does not reflect the pathological state in human cirrhotic liver, in vitro examination utilizing the cultured rat HSC can be a efficient tool for the assessment of the role of Grb10 in the mechanism of hepatic fibrosis in future.

观察Grb10在人病理性肝硬化肝组织及肝损伤动物模型中的表达。二甲基亚硝胺（DMN）给药和CCI4给药小鼠模型根据先前报道的方案构建，但由于其高致死率和肝细胞损伤而非门脉纤维化，因此未用于后续实验。然后对胆总管结扎（CBDL）模型进行Grb10免疫组化染色，但未见明显染色。RT-PCR检测CBDL小鼠和正常成年小鼠肝组织mRNA表达，Grb10协同表达。这一结果与之前报道的Grb10在成年小鼠肝脏中的表达与胚胎中的表达相比非常低有所不同。另一方面，在Western blot中，与胎儿和CBDL小鼠肝脏相比，成年小鼠肝脏中的蛋白表达非常低。提取大鼠肝实质细胞和肝星状细胞（HSC），观察HSC的成纤维转化情况，采用RT-PCR检测Grb10的表达。结果，在肌成纤维细胞（HSC）中检测到mRNA的表达，而在肝细胞中表达极低。此外，在dmn处理的SD大鼠损伤的肝组织中，mRNA的表达与在人肝硬化中发现的定位相同。虽然这并不能反映人肝硬化的病理状态，但利用培养的大鼠HSC进行体外检查，可以成为未来评估Grb10在肝纤维化机制中的作用的有效工具。