| Co-Investigator(Kenkyū-buntansha) |
OKAWA Hiroyuki CHUGAI Pharmaceutical Co., LTD.
HIGASHI Sayumi CHUGAI Pharmaceutical Co., LTD.
HATA Jun-ichi National Research Institute for Child Health and Development, General Director, 研究所, 研究所長 (90051614)
OKITA Hazime KEIO UNIVERSITY SCHOOL OF MEDICINE, Department of Pathology, Research Assistant, 医学部, 助手 (50317260)
FUKUMA Mariko KEIO UNIVERSITY SCHOOL OF MEDICINE, Department of Pathology, Research Assistant, 医学部, 助手 (60101995)
東佐 由美 中外製薬(株), 育成研究センター, (研究職)
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| Budget Amount *help |
¥12,000,000 (Direct Cost: ¥12,000,000)
Fiscal Year 2002: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2001: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2000: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1999: ¥4,400,000 (Direct Cost: ¥4,400,000)
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| Research Abstract |
KUSA/A1 cells were originally isolated as cells which induce hematopoiesis in vivo. When cultured in monolayer, the cells were spindle-shaped in the growth phase and after confluence. Extracellular matrix positive for von Kossa stain appeared in cell culture and increased after confluence. Ultrastructurally, the matrix was electron dense and was clearly produced by the cells. We analyzed the cell surface markers on the clonal KUSA/A1 cells by flow cytometry to better characterize these cells. The cells were found to be strongly positive (more than 10-fold greater than the isotype control) for Sca-1, CD44, Ly-6C and CD140, weakly positive for CD29, and negative for c-kit, Flk-1, CD14, CD31, CD34, CD41, CD45, CD49b, CD49d, CD54, CD90, CD102, CD105, CD106, CD144 and Ly-6G. We then analyzed growth curve, ALP activity, in vitro calcification, osteocalcin (bone gla protein) release, and response to PTH, in comparison with MC3T3-E1 cells. KUSA/A1 proliferation, which was measured by DNA content, was equivalent to that of MC3T3-E1 cells. ALP activity of KUSA/A1 cells in growth phase was approximately 10-fold higher than in MC3T3-E1 cells. In vitro calcification, which steadily increased both before and after confluence, was approximately 100-fold higher after the confluent stage in KUSA/A1 cells than in MC3T3-E1 cells. Osteocalcin release into culture media peaked on day 5 in KUSA/A1 cells and was 2 to 3-fold higher than in MC3T3-E1 cells. PTH response, measured as cAMP production, gradually decreased during the culture period in KUSA/A1 cells, but increased in MC3T3-E1 cells. This response to PTH did not require any induction and was independent of culture conditions.
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