| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1990: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1989: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1988: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
Several reports have suggested that proteoglycans produced by stromal cells in a hemopoietic organ are involved in a regulation of hemopoiesis. It has not been, however, elucidated what and how proteoglycans do that. Using a clonal cell line of murine preadipocyte, MC3T3-G2/PA6, which has been demonstrated to have an ability to support self-renewal and differentiation of hemopoietic stem cells in vitro, we have studied the nature of the proteoglycans produced by the cells and their roles on hemopoiesis. The results obtained revealed the followings : The cells produced four molecular species of proteoglycans with different molecular weights. The three of them were associated with the cell layer and the one was released into the medium. The major glycosaminoglycans of the formers were heparan sulfate, whereas that of the latter was chondroitin sulfate. The cells cultured in the presence of beta-D-xyloside, an artificial initiator of glycosaminoglycan elongation, stimulated the ability to
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support hemopoiesis in vitro.
To elucidate which type of the proteoglycans were involved in supporting hemopoiesis in this system, we have tried to separate the proteoglycans on their biochemical properties. The proteoglycans were applied to a column of Octyo-Sepharose CL-4B and the column was eluted with a linear gradient-concentration of Triton X-100 (0 - 0.5%). The whole proteoglycans released into the medium passed through the column, indicating that they have no hydrophobic domain in their core proteins. On the contrary, the cell layer-proteoglycans were separated into three fraction, i. e., 55% of them passed through the column, 15% of them bound and eluted with 0.03% Triton X-100, and 25% eluted with 0.2% Triton X-100. These results strongly suggested that PA6 cells produced at least two types of proteohlycans, nanely, basement membrare-type proteohlycans (0.03% Triton X-100 fraction) and cell membrane-type proteoglycans (0.2% Triton X-100).
We are now trying to produce monoclonal antibodies against these proteoglycans. Their roles on hemopoiesis will be demonstrated by using these tools. Less
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