| 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.
|
