Project/Area Number |
07557354
|
Research Category |
Grant-in-Aid for Scientific Research (A)
|
Allocation Type | Single-year Grants |
Section | 展開研究 |
Research Field |
内分泌・代謝学
|
Research Institution | Nagasaki University |
Principal Investigator |
KONDO Takahito Nagasaki University, School of Medicine, Professor, 医学部, 教授 (00158908)
|
Co-Investigator(Kenkyū-buntansha) |
TOMONAGA Masao Nagasaki University, School of Medicine, Professor, 医学部, 教授 (40100854)
YAMASHITA Shunichi Nagasaki University, School of Medicine, Professor, 医学部, 教授 (30200679)
長瀧 重信 長崎大学, 医学部, 教授 (70010311)
|
Project Period (FY) |
1995 – 1997
|
Project Status |
Completed (Fiscal Year 1997)
|
Budget Amount *help |
¥8,500,000 (Direct Cost: ¥8,500,000)
Fiscal Year 1997: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 1996: ¥4,500,000 (Direct Cost: ¥4,500,000)
|
Keywords | gamma-glutamylcysteine / glutathione / ribozyme / IDDM / beta-Cell / redox / NF-_kB / TNF-alpha / NF-κB / マウスβ細胞 / β-GCS / サイトカイン |
Research Abstract |
Development of gene therapy for IDDM addressing regration of pancreatic beta-cell death. We used mouse pancreatic beta-cell lines, Min-6 and beta-TC as materials. We estimated antioxidant activities, insulin secretion, expression of preproinsulin gene, and analyzed the preproinsulin gene promoter. beta-cell damage was estimated as DNA damage induced by the treatment of these cells with TNF-alpha and IL- 1beta. To regulate the antioxidant activity and redox, we cloned the gene of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme for the glutathione (GSH) synthesis, and analyzed the 5'-flanking region of gamma-GCS gene. To suppress the expression of gamma-GCS,beta-cells were transfected with antisense codon of gamma-GCS constructed with ribozyme. Results 1. Levels of GSH and activities of GSH related enzymes decreased by 90% in Min-6 and beta-TC cells compared to other mouse tissues cells, suggesting beta-cells are feasible to oxidative stress and possess less redox activity. 2. The insulin secretion was down-regulated by GSH. 3. The expression of preproinsulin was not regulated by GSH. 4. GSH protected beta-cell damage by cytokines. 5. Transfection of anti-gamma-GCS-ribozyme caused of increase in insulin-secretion. 6. These data suggest low concentration of GSH in beta-cells is essential for the insulin secretion while sensitive to cellular damage by oxidants. Collectively, new gene therapy targeting the suppression of GSH synthesis thought to be effective to IDDM.
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