| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2001: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
We investigated whether neuronal stem cells produce interleukin-1 receptor antagonist (IL-1ra) in vitro by ELISA. Neuronal stem cells were prepared from the forebrain of embryos (Sprague-Dawley rat, E12), and were cultured in DMEM/F12 containing B27 supplement and growth factors, both 20 ng/ml of FGF-2 and 20 ng/ml of EGF, under a condition of 5% CO2 at 37 ℃. A cluster of cells obtained to be consequently larger than 50 m diameter was defined as a neurosphere. A neurosphere was dissociated by pippeting gently and was continued culturing for 10 days to obtain the next generation of neurospheres. Afterwards, each neurosphere was transferred into a well of 96-well culture plate containing 100 l of culture medium. Twenty microlitter of medium was collected every 24 hours, followed by addition of the same volume of fresh medium, for 4 days. The sample was stored at -80 ℃ until measurement of IL-1ra. After the last collection of the medium, number of cells alive in the neurosphere was counted. Amounts of IL-1ra were detected by using an ELISA kit, whose detection limit was 12 pg/ml. Production of IL-1ra was found in 6.7% neurosphere of 4-days culture. A concentration of IL-1ra was 1.58 pg/106 cells. It is not clear whether IL-1ra-producing cells were neuronal stem cells, because a neurosphere consists of not only neuronal stem cells but also progenitor cells. Number of neurospheres which were detected producing IL-1ra increased as the passage of culture up, in spite of uncharged concentration of IL-1ra. Since a neurosphere should be generated from a neuronal stem cell, this may be explained by considering that IL-1ra-producing neuronal stem cells could survive to generate the neurosphere without being selected out. Results suggest that a neuronal stem cell has a possible productivity of IL-1ra, and that IL-1ra may play a role in surviving the cell.
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