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Effect of hepatocyte growth factor on liver regeneration and damage of gepatocytes

肝细胞生长因子对肝再生及肝细胞损伤的影响

基本信息

批准号：
03671051
负责人：
GOHDA Eiichi
金额：
$ 1.34万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03671051/
关键词：
Hepatocyte growth factor Hepatocytes in primary culture Hepatocyte damage Liver regeneration Hepatoma cells

项目摘要

We found previously that mouse hepatocyte growth factor (mHGF) in serum and liver markedly increased prior to liver regeneration in carbon tetrachloride-treated mice. mHGF was then purified about 280,000-fold from the liver supernatant of mice treated with the hepatotoxin and was characterized. It is composed of two peptide chains with molecular masses of 62 and 31 kDa, linked by a disulfide bond(s). Its physicochemical and biological properties are quite similar to those of hHGF. In this study, we found that homogenizing the damaged liver with 1.7 M NaCl (pH 7.4) in phosphate-buffered saline (PBS) extracted 5-10-fold more mHGF than homogenization of the liver with 0.25 M sucrose containing 20 mM Hepes (pH 7.4). mHGF, extracted by this modified method, was purified by our four-step purification method. We next examined whether D-galactosamine-induced damage of rat hepatocytes in primary culture is prevented by adding mHGF or hHGF to the hepatocyte cultures. mHGF or hHGF did not diminish the hepatotoxicity of D-galactosamine at doses of 10-30 ng/ml, which induce DNA synthesis in hepatocytes. Effect of hHGF on the growth of various cultured cells was next investigated. It stimulated proliferation of HuH-6 Clone 5 human hepatoblasstoma cells, but did not that of HLE, HuH-7, and PLC/PRF/5 human hepatoma cell lines. The growth of Hep G2, another human hepatoblastoma cell line, was inhibited by hHGF. Studies on other hepatoma and hepatoblastoma cell lines are required to determine whether hepatoblastoma, and not hepatoma cells are responsive to hHGF. It has not been clear whether hHGF affect the growth of fibroblasts, but in this study we demonstrated significant mitogenic activity of hHGF for human skin fibroblasts. We reported previously that the fibroblasts secreted a small but detectable amount of hHGF. Furhter studies are required to answer the question of whether proliferation of human skin fibroblasts is stimulated by hHGF in an autocrine manner.

我们以前发现，小鼠肝细胞生长因子（mHGF）在血清和肝脏显着增加之前，四氯化碳处理的小鼠肝再生。然后从用肝毒素处理的小鼠的肝上清液中纯化mHGF约280，000倍，并进行表征。由分子量为62和31 kDa的两条肽链组成，通过二硫键连接。其理化和生物学性质与hHGF非常相似。在这项研究中，我们发现用磷酸盐缓冲盐水（PBS）中的1.7 M NaCl（pH 7.4）浸泡受损的肝脏比用含有20 mM Hepes的0.25 M蔗糖（pH 7.4）匀浆肝脏提取的mHGF多5-10倍。通过这种改进的方法提取的mHGF通过我们的四步纯化方法纯化。我们接下来检查了在原代培养的大鼠肝细胞中D-半乳糖胺诱导的损伤是否可以通过向肝细胞培养物中加入mHGF或hHGF来预防。mHGF或hHGF在10-30 ng/ml的剂量下不降低D-半乳糖胺的肝毒性，其诱导肝细胞中的DNA合成。接下来研究hHGF对各种培养细胞生长的影响。它刺激HuH-6克隆5人肝母细胞瘤细胞的增殖，但不刺激HLE、HuH-7和PLC/PRF/5人肝癌细胞系的增殖。人肝母细胞瘤细胞系Hep G2的生长受到hHGF的抑制。需要对其他肝癌和肝母细胞瘤细胞系进行研究，以确定肝母细胞瘤而不是肝癌细胞是否对hHGF有反应。目前尚不清楚hHGF是否影响成纤维细胞的生长，但在这项研究中，我们证明了hHGF对人皮肤成纤维细胞具有显着的促有丝分裂活性。我们以前报道过成纤维细胞分泌少量但可检测到的hHGF。hHGF是否以自分泌方式刺激人皮肤成纤维细胞增殖，有待进一步研究。