|Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2001: ¥1,800,000 (Direct Cost: ¥1,800,000)
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.