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REGULATION OF PROMER OF TGF-β/SMAD-TARGET GENE FOR ATTENUATION OF RENAL FIBROSIS

TGF-β/SMAD 靶基因启动子的调节以减轻肾纤维化

基本信息

批准号：
13671132
负责人：
TAMAKI Kiyoshi
金额：
$ 2.3万
依托单位：
KURUME UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671132/
关键词：
Growth factor TGF-β Smad renal fibrosis Smad binding element Lux GFP

项目摘要

TGF-β/Smad-target gene such as plasminogen activator inhibitor has Smad-binding element (SEE) in its promoter region. We generated artificial plasmid consisted of luciferase gene with SBE (SBE-Lux) in its promoter region. Using this plasmid, we have done experiments as follows. Cultured mesangial cells or smooth muscles cells was transfected with the plasmid and the cultured cells were treated with TGF-β or the substances, which are shown to induce the production of TGF-β. TGF-β increased the activity of SBE-Lux in dose-dependent manner. Angiotension II and Advanced glycation end product (AGE) also stimulated SBE-Lux activity. The activity was blocked by the presence of neutralizing anti-TGF-β antibody.Next, we generated artificial plasmid consisted of GFP (green fluorescence product) gene with SBE (SBE-GFP) in its promoter region. The cells transfected with the construct showed TGF-β-induced green color in its cytoplasma. Thus, the cell showing green fluolorescence was considered as TGF-β-target-cells. However, the cultured cells, which were transfected with the construct and treated with Angiotension II or AGE, have not shown green color enough to be identified. In addition, the tissues, which were transfectected with SBE-GFP, have not shown the green color induced by sclerotic damage. High concentration of TGF-β more than 10^<-2>ng/ml is necessary to induce the green fluolorescence in the transfected cells. Such high concentration of TGF-β was not considered to exist in the physiological or pathological condition. Thus, more sensitive artificial promoter is necessary to detect TGF-β activity.

TGF-β/Smad靶基因如纤溶酶原激活物抑制剂（plasminogen activator inhibitor，PAI）在其启动子区含有Smad结合元件（SEE）。我们构建了荧光素酶基因启动子区带有SBE（SBE-Lux）的人工质粒。利用该质粒，我们进行了如下实验。用质粒转染培养的系膜细胞或平滑肌细胞，并用TGF-β或显示诱导TGF-β产生的物质处理培养的细胞。TGF-β呈剂量依赖性地增加SBE-Lux的活性。血管紧张素II和晚期糖基化终产物（AGE）也刺激SBE-Lux活性。然后，我们构建了由GFP（绿色荧光产物）基因和启动子区的SBE（SBE-GFP）组成的人工质粒。转染该构建体的细胞在其胞浆中显示TGF-β诱导的绿色颜色。因此，显示绿色荧光的细胞被认为是TGF-β-靶细胞。然而，用构建体转染并用血管紧张素II或AGE处理的培养细胞没有显示出足以被鉴定的绿色。此外，用SBE-GFP转染的组织没有显示出由皮肤损伤诱导的绿色颜色。高浓度的TGF-β（> 10 μ <-2>ng/ml）是诱导转染细胞产生绿色荧光所必需的。认为在生理或病理条件下不存在如此高浓度的TGF-β。因此，需要更敏感的人工启动子来检测TGF-β活性。