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Development of Large Scale Expression, Preparation, and Highly Sensitive Immunoassay Methods for Hepatocyte Growth Factor

肝细胞生长因子大规模表达、制备和高灵敏免疫分析方法的开发

基本信息

批准号：
03558020
负责人：
NAKAMURA Toshikazu
金额：
$ 10.18万
依托单位：
Osaka Univrsity (1992)Kyushu University (1991)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
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-03558020/
关键词：
HGF Liver Rebeneration Renal Regeneration Anti Hepatitic Action ELISA RIA 肝細胞増殖因子肝炎増殖因子

项目摘要

Hepatocyte growth factor (HGF) has mitogenic, and morphogenic activities for various types of epithelial cells, including mature hepatocytes. In 1989, we succeeded in molecular cloning of HGFcDNA and elucidated primary structure of HGF. Although HGF has been considered to act as hepatotrophic factor for liver regeneration, we recently found that HGF acts as renotropic act as renal regeneration. In the present study, we have succeeded in the development of large scale expression and preparation of recombinant HGF, and highly sensitive immunoassay for HGF, as follows:(1) The expression vector for human HGF was transfected in mammalian cells and we obtained cell line which produces recombinant human HGF in extremely large scale (10 mg/l). Thus method for large scale preparation of HGF has been established. (2) Using polyclonal and monoclonal antibodies to human HGF, we established highly sensitive immunoassay method for human HGF. (3) We also established methods for large scale preparation of rat HGF and its highly sensitive immunoassay. (4) We found HGF levels in sera of patients with hepatitis, partial hepatectomy, acute renal failure, renal carcinoma, lung cancer, and pneumonia are much higher than those in healthy persons. (5) We obtained direct evidence that HGF acts as hepatotrophic and renotropic factor, when recombinant HGF was intravenously injected into experimental animals subjected to various types of hepatic of renal injuries, HGF remarkably stimulated hepatic and renal regeneration. All these results indicate that HGF may prove to be a an useful treatment to accelerate and renol regeneration and also to be an diagnostic marker for various organ diseases. Therefore we expect that HGF will make great contributions not only for basic sciences but also for clinical medicine.

肝细胞生长因子（HGF）对各种类型的上皮细胞（包括成熟肝细胞）具有促有丝分裂和形态发生活性。1989年，我们成功地克隆了HGF cDNA，并阐明了HGF的一级结构。虽然肝细胞生长因子一直被认为是肝再生的肝营养因子，但最近我们发现肝细胞生长因子在肾再生中也有促肾功能的作用。本研究成功地建立了重组人HGF的大规模表达、制备和高灵敏度的免疫检测方法，具体如下：（1）将人HGF表达载体转染哺乳动物细胞，获得了能大量（10 mg/l）产生重组人HGF的细胞株。从而建立了大规模制备HGF的方法。(2)利用抗人肝细胞生长因子的多克隆抗体和单克隆抗体，建立了高灵敏度的人肝细胞生长因子免疫分析方法。(3)建立了大鼠肝细胞生长因子的大规模制备方法和高灵敏度的免疫分析方法。(4)发现肝炎、肝部分切除、急性肾功能衰竭、肾癌、肺癌、肺炎患者血清中HGF水平明显高于健康人。(5)我们获得了肝细胞生长因子（HGF）具有促肝、肾功能的直接证据，将重组HGF静脉注射到各种类型肝、肾损伤的实验动物体内，可明显促进肝、肾的再生。所有这些结果表明，肝细胞生长因子可能被证明是一个有用的治疗，以加速和肾再生，也是一个诊断指标，为各种器官疾病。因此，我们期待HGF不仅在基础科学上，而且在临床医学上做出巨大的贡献。