| Project/Area Number |
10480244
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biomedical engineering/Biological material science
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| Research Institution | Osaka University |
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Principal Investigator |
YOSHIDA Toshiomi IC Biotech., Osaka University, Professor, 生物工学国際交流センター, 教授 (00029290)
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| Co-Investigator(Kenkyū-buntansha) |
SAWA Yoshiki Faculty of Medicine, Osaka University, lecturer, 医学部・第一外科, 講師 (00243220)
TAKAGI Mutsumi IC Biotech., Osaka University, Associate Professor, 生物工学国際交流センター, 助教授 (20263212)
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| Project Period (FY) |
1998 – 2000
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥11,700,000 (Direct Cost: ¥11,700,000)
Fiscal Year 2000: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1999: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1998: ¥8,900,000 (Direct Cost: ¥8,900,000)
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| Keywords | bone marrow cells / progenitors / stem cells / stromal cells / cvtokine / porous carriers / genetic engineering / cultivation |
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| Research Abstract |
Murine stromal cell lines of ST2 and SR-4987 transfected with the gene coding interleukin-3 (IL-3) employing adenovirus vector produced IL-3, but dead within 1 week.
In the next step, transfection employing plasmid vector instead of virus was studied. The gene coding murine stem cell factor (mSCF) in pCAmKL(RDB1528) was combined to a plasmid vector for eukaryote of pcDNA3. 1/Hygro(+) (InvitrogenCo.) and transfected to ST2 cells by means of lipofection method. The tranformants were selected by the incubation together with RPMI1640 medium containing hygromycin. Murine bone marrow hematopoietic cells (5 x 10^5 cells/ml) were cultivated on the layer of the transformed ST2 cells (strain DK1-2, DK2-2, DK2-3) (5 x 10^3 cells/cm^2) in a T-flask (8.3cm^2) using McCoy's 5A medium (5 ml) at 33℃ under the atmosphere of 5% CO_2. Half of culture supernatant containing hematipoietic cells was harvested weekly and the same volume of fresh medium was added. Hematopoietic cell concentration and the content of progenitor cells were analyzed for each sample.
There was no marked difference in SCF concentration between original strain and transformed strains. The contents of CFU-G/M/GM in the co-culture with all transformed cells were higher than that with original strain. The contents of erythroid progenitor in the culture with DK2-2 was higher than the original and those with DK1-2 and DK2-3 were lower. The content of CFU-Mix which was the most primitive progenitor for DK2-3 was higher and those for DK1-2 and DK2-2 were lower than that with original strain.
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