| Project/Area Number |
12358015
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Biomedical engineering/Biological material science
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| Research Institution | Tokyo Institute of Technology |
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Principal Investigator |
AIZAWA Masuo Tokyo Institute of Technology, Professor, 大学院生命理工学研究科, 教授 (00016742)
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| Co-Investigator(Kenkyū-buntansha) |
YANAGIDA Yasuko Tokyo Institute of Technology, Lecturer, 大学院生命理工学研究科, 講師 (10282849)
TAJIMA Naoko Jikei University, School of Medicine, Professor, 医学部, 教授 (70112836)
SASAKI Takashi Jikei University, School of Medicine, Lecturer, 医学部, 講師 (90205849)
OHNO Tsuneya Jikei University, School of Medicine, Professor, 所長 (60147288)
NAKAMURA Mariko Jikei University, School of Medicine, Associate Professor, 医学部, 助教授 (40237433)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥42,720,000 (Direct Cost: ¥38,400,000、Indirect Cost: ¥4,320,000)
Fiscal Year 2001: ¥18,720,000 (Direct Cost: ¥14,400,000、Indirect Cost: ¥4,320,000)
Fiscal Year 2000: ¥24,000,000 (Direct Cost: ¥24,000,000)
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| Keywords | Insulin / Gee expression / Electrical stimulation / hsp 70 promoter / furin / Cell device / Myoblasts / preadipocytes / furin / 筋芽細胞 / レトロウイルス / HSPプロモーター / 3T3L1細胞 / エンカプシレーション |
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| Research Abstract |
The research group in Jikei University has focused on generatio of novel cell lines that secrete insulin. Murine preadipocytes and myoblasts were engineered by the modified insulin cDNA with a recombinant retroviral vector whose proinsulin could be processed by ubiquitous convertase furin. Human insulin was detected by a human-specific immunoassay in culture media, and also by immunocytostaining.
Tokyo Tech. research group have discovered that the 10 Hz frequency 0 to 0.3 V sine wave potential of electric stimulation one of the very slight physical stresses could induce the promoter activation of heat shock 70 (hsp70) promoter. Using this property, we constructed the expression vector containing engineered insulin cDNA under the control of hsp70 promoter. When transfected C2C12 myoblast cells were cultured under heat shock condition or electric stimulation the insulin contraining cultured medium was increased at 24 hour after stimulation.
To accomplish our objectives "In vitro insulin production and secretion system", we have designed a basic diagram of the cell device to appropriate production of insulin by transformants celles in an environment of separatio from host immune systems and tried to produce the prototype by coordination with the scientists of In Vitrogen. In Vitro testing on cell chamber dummies for mid term stability, insulin passage, antibody passage and glucose passage before and after cell growth in the internal chamber. Furthermore, to quantitative the amount of insulin produced precisely, we evaluated a micro assay to quantificate the amount of insulin molecules by ELISA and BIACORE. When the cells were transplanted to diabetic mice with immunoisolating chamber, glucose level was recovered to near normal, demonstrating the biological potency of transplantation of the engineered cells. In addition to this, we found that insulin secretion from differentiated cells showed ten times larger than that from undifferentiated cells.
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