| Project/Area Number |
06557061
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
内分泌・代謝学
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| Research Institution | KUMAMOTO UNIVERSITY |
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Principal Investigator |
SHICHIRI Motoaki Kumamoto University, School of Medicine, Professor, 医学部, 教授 (00028515)
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| Co-Investigator(Kenkyū-buntansha) |
MIYATA Takao Kumamoto University, School of Medicine, Research Associate, 医学部, 助手 (70244118)
SAKAKIDA Michiharu Kumamoto University, Hospital, Research Associate, 医学部・附属病院, 助手 (50170577)
ARAKI Eiich Kumamoto University, School of Medicine, Research Associate, 医学部, 助手 (10253733)
KISHIKAWA Hideki Kumamoto University, School of Medicine, Lecturer, 医学部, 講師 (30161441)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥15,200,000 (Direct Cost: ¥15,200,000)
Fiscal Year 1995: ¥7,200,000 (Direct Cost: ¥7,200,000)
Fiscal Year 1994: ¥8,000,000 (Direct Cost: ¥8,000,000)
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| Keywords | Human insulin gene / CHO cells / 3T12-3 fibroblasts / AtT20 cells / MIN6 cells / Glucose transporter type 2 gene / Glucokinase gene / CHO細胞 / AtT20細胞 / インスリン遺伝子発現細胞 / 線維芽細胞系腫瘍細胞 / HPLC / インスリン / プロインスリン |
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| Research Abstract |
In this study, we transfected human insulin gene in some eukaryotic cells ; CHO cells, 3T12-3 fibroblasts, pituitary ACTH secreting tumor cells (AtT20) or murine insulinoma cells (MIN6 cells). And their characteristics were evaluated in vitro and in vivo.
Expression of human insulin gene in these cells was confirmed by Northern blot analysis. Radioimmunoassay and HPLC revealed that human insulin gene transfected CHO cells and 3T12-3 fibroblasts secreted proinsulin into culture medium, whereas human insulin gene transfected AtT20 cells and MIN6 cells did insulin. In addition, all of these human insulin gene transfected cells induced hypoglycemia, when these cells were transplanted into nude mouse. Nude mouse died at 15 days after the transplantation of insulin gene transfected CHO cells and at 24 hours after the transplantation of insulin gene transfected MIN6 cells.
Human insulin gene transfected MIN6 cells showed glucose concentration-dependent insulin secretion in culture media, but human insulin gene transfected AtT20 cells did not. Also, in the perifusion experiment, human insulin gene transfected MIN6 cells revealed biphasic insulin secretion in responce to glucose, but human insulin gene transfected AtT20 cells did not. On the other hand, AtT20 cell transfected with insulin gene, glucose transporter type 2 gene and glucokinase gene showed the glucose concentration-dependent insulin secretion in culture media, although it did not reveal biphasic insulin secretion in responce to glucose in the perifusion experiment.
The construction of pancreatic B cell functions gene-biotechnologically in eukaryotic cells might imply not only the understanding of unknown mechanisms of glucose sensing system, signal transduction system and insulin secretion in pancreatic B cell, but also the possible-model of insulin gene-therapy in diabetes treatment.
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