| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1993: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1992: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
We investigated the action of neurotrophic factors to rat oligodendrocytes, oligodendrocyte progenitor cells in vitro. Brain cells from newborn or 1 week-old rats were trypsinized. Cells were cultured in 2.5% FCS medium overnight. At 2DIV, the coverslips are transferred to the well in which 5 mu g/ml insulin and 0.5% FCS are contained. In the culture, FGF(10ng/ml), EGF(30ng/ml), PDGF(10ng/ml), endothelin-3(10-8M), retinoic acid (1-50 mu M) were added and cultured for 4-9days. 24hours before immunofluorescene, (oligodendrocyte ; mouse anti-galactocerebroside antibody, 0-2A progenitor ; A2B5) 10 mu M of bromodexyuridine (BrdU) was added. Under fluorescence microscope, antigen-positive cells were counted.
Insulin was necessary for survival of cells. FGF enhanced the mitotic activity of oligodendrocytes. But it showed weaker action on 1-2A progenitor cells. EGF enhanced mitotic activity of oligodendrocytes, but its action was not shown in newborn cells, and it showed the enhanced mitotic activity in 1 week-old brain cells. PDGF exerted enhanced mitotic activity to oligodendrocytes and progenitor cells. Endothelin-3 showed enhanced mitotic activity in early cultures of 0-2A progenitor cells and oligodendrocytes but in longer culture, these cells became degenerated and astrocyte proliferation was observed making bundles. Retinoic acid was toxic to both cells.
Oligodendrocytes were cultured from 4 week-old rat brain isolated by Percoll density gradient. In medium with 0.5%FCS and insulin, oligodendrocytes extended the processes but thereafter degenerated with fragmentation of processes from the cell body. FGF,PDGF and EGF had no mitogenic effect on mature oligodendrocytes. However these neurotrophic factors enhanced the survival of oligodendrocytes and seemed to be the survival factors of oligodendrocytes.
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