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The Study of the Hepatotrophic Factor in Sera of Patients with Liver Disease. -- its clinical significance and purification --

肝病患者血清中肝营养因子的研究。

基本信息

批准号：
60570297
负责人：
HIRONO Shuichi
金额：
$ 1.09万
依托单位：
Kagoshima 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：
1985
资助国家：
日本
起止时间：
1985 至 1986
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-60570297/
关键词：
Hepatotrophic Factor Fulminant Hepatic Failure Primary Culture of Adult Rat Hepatocytes DNA Synthesis Human Hepatocyte Growth Factor hHGF 精製

项目摘要

According to the initial plan, we performed the study on the human hepatocyte growth factor(hHGF) and obtained the following results.(1)The effect of sera from patients with various liver diseases on liver cell growth was investigated by use of adult rat liver cells in primary culture. Sera from patients with chronic hepatitis or liver cirrhosis had no or slight growth- promoting activity, respectively, compared with that of normal human sera. Sixteen of seventeen sera from patients with fulminant hepatic failure showed marked stimulatory activity on DNA synthesis of about 16 times an average value of normal human sera. The growth- promoting activity was confirmed by showing that the patients' serum increased the labeling index with [ <^3H> ]thymidine and total number of nuclei in hepatocyte cultures.(2)Appearance of hHGF in sera of patents with fulminant hepatic failure seems to be related to their coma grade. Its level increased when the patient fell into a state of more advanced coma and decreased immediately in the recovery stage.(3)Plasma from patients with fulminant hepatic failure obtained during plasma exchange therapy, like their serum,demonstrated marked stimulatory activity on DNA, synthesis in cultured rat hepatocytes. The activity of pooled active fractions, obtained by gel filtration of the heated plasma, was lost completely on heat treatment at 80 ゜C for 10 min. or on treatment with trypsin or chymotrypsin, which suggests that it was due to a protein.HHGF was purified about 10,000-fold from heated plasma of a patient by ammonium sulfate precipitation and chromatographies on Affi-Gel Blue and heparin-Sepharose. The molecular weight of hHGF was about 85,000 as determined by SDS-PAGE.

按照初步计划，我们对人肝细胞生长因子（hHGF）进行了研究，得到了以下结果：（1）利用原代培养的成年大鼠肝细胞，研究不同肝病患者血清对肝细胞生长的影响。与正常人血清相比，慢性肝炎或肝硬化患者的血清分别没有或仅有轻微的生长促进活性。来自暴发性肝衰竭患者的 17 份血清中有 16 份显示出对 DNA 合成的显着刺激活性，约为正常人血清平均值的 16 倍。通过显示患者血清中[<^3H>]胸苷标记指数和肝细胞培养物中细胞核总数的增加证实了其促生长活性。(2)暴发性肝衰竭患者血清中hHGF的出现似乎与其昏迷等级有关。当患者陷入更严重的昏迷状态时，其水平升高，在恢复阶段立即下降。(3)血浆置换治疗期间获得的暴发性肝衰竭患者的血浆，与血清一样，对培养的大鼠肝细胞中的DNA合成具有明显的刺激活性。通过对加热血浆进行凝胶过滤获得的合并活性组分的活性在 80°C 热处理 10 分钟后完全丧失。或用胰蛋白酶或胰凝乳蛋白酶治疗，这表明它是由蛋白质引起的。通过硫酸铵沉淀和 Affi-Gel Blue 和肝素琼脂糖上的色谱法，将 HHGF 从患者的加热血浆中纯化约 10,000 倍。通过SDS-PAGE测定hHGF的分子量约为85,000。