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The tissue distribution of collagen-binding heat shock protein, HSP47, in fibrotic diseases.

纤维化疾病中胶原蛋白结合热休克蛋白 HSP47 的组织分布。

基本信息

批准号：
02670148
负责人：
SAGA Shinsuke
金额：
$ 1.47万
依托单位：
Nagoya 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：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02670148/
关键词：
Heat shock protein Collagen Collagen-binding Antibody Fibrotic diseases

项目摘要

Heat shock protein of molecular weight 47, 000 D, HSP47, binds to native and denatured collagen including type I, type III, or type IV. HSP47 is supposed to be implicated in the transportation of procollagen molecules from ER to Golgi. In order to determine immunohistochemically the distribution of HSP47 in liver or lung from the patients with fibrotic diseases, polyclonal and monoclonal antibodies against chicken HSP47 were used. Howerer, in the fixed mammalian tissues such antidobies were not able to atain HSP47 because of the disruption of the epitopes by fixation. Therefore, in order to get the antibody that react to human HSP47 even in the fixed tissues, human HSP47 was partially purified from NP-40 extract of human embryonal fibroblasts cultured in at least 10 roller bottles by gelatin affinity chromatography or immunoaffinity chromatography. It was very difficult to get further purified HSP47 because human HSP47 in the partially purified fraction was extremely unstable and easily degraded. This difficulty disturbed the production of the specific polyclonal or monoclonal antibody against human HSP47. Thus, immunohistochemical study for the distribution of HSP47 in the fibrotic diseases was not proceeded.The trial of in situ hybridization with cDNA probe of mouse HSP47 in the section of liver or lung tissue of the fibrotic diseases failed to determine the distribution of mRNA for HSP47.

分子量为47，000 D的热休克蛋白（HSP 47）与天然和变性胶原蛋白（包括I型、III型或IV型）结合。HSP 47被认为与前胶原分子从ER到高尔基体的运输有关。为了用免疫化学方法检测HSP 47在纤维化疾病患者肝、肺组织中的分布，本研究采用了鸡HSP 47多克隆抗体和单克隆抗体。然而，在固定的哺乳动物组织中，由于固定破坏了表位，这些抗体不能抑制HSP 47。因此，为了得到即使在固定的组织中也能与人HSP 47反应的抗体，通过明胶亲和层析或免疫亲和层析从至少10个滚瓶中培养的人胚胎成纤维细胞的NP-40提取物中部分纯化人HSP 47。由于部分纯化组分中的人HSP 47极不稳定且易降解，因此很难进一步纯化。这种困难干扰了针对人HSP 47的特异性多克隆或单克隆抗体的产生。因此，未进行HSP 47在纤维化疾病中分布的免疫组织化学研究，用小鼠HSP 47的cDNA探针在纤维化疾病的肝或肺组织切片中进行原位杂交试验，未能确定HSP 47的mRNA分布。