|Budget Amount *help
¥2,600,000 (Direct Cost : ¥2,600,000)
Fiscal Year 1999 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1998 : ¥1,800,000 (Direct Cost : ¥1,800,000)
(1) Differentiation capacity of TBR 31-2 cells
To study the differentiation capacity of TBR 31-2 cells, the m-RNA expression of differentiation factor, osteoblast core binding factor 1and differentiation phenotypes such as alkaline phosphatase, type I collagen, osteocalcin, osteopontin, PTH receptor and vitamin D receptor were examined by reverse transcription-polymerase chain reaction. Expression of these proteins was only observed in the stage of cell differentiation. In addition, the increase of m-RNA expression level in differentiation factor, peroxisome proliferator-activated receptor γ and differentiation markers such as lipoprotein lipase and adipsin were also observed. These observations suggest that TBR 31-2 cells show bipotential characters, differentiation toward adipocytes and osteoblasts.
(2) Purification and identification of GPI-anchor proteins in TBR 31-2 cells
After biotinylation of peripheral proteins present on the plasma membranes of TBR 31-2 cells, GPI-anchor proteins were released by the treatment of PI-PLC. Solubilized GPI-anchor proteins were separated by SDS-PAGE, blotted to nitrocellulose membrane, and then analyzed chemiluminescence using peroxidase-conjugated streptoavidin. Four proteins having molecular weights of 120, 112, 96 and 60kDa were specifically solubilized by the action of PI-PLC.