| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2003: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
Blood and urine analysis : Although the level of serum calcium remained mostly within the normal range for both groups, there was a significant increase in the level of urinary Ca for the FK506 treated group from Week 1 of administration. The level of blood osteocalcin for the FK506 treated group at Week 1 of administration was significantly higher than that for the control group, but there was no significant difference after that. Throughout the administration period, the sum of urinary PYD and Dpd for the FK506 treated group was significantly higher. Of the various bone resorption factors, the level of blood PTH for the FK506 treated group was significantly higher than that for the control group at Week 3 of administration and later.
Cell culture : After acclimatization for a week, mice were randomly divided into two groups and were intraperitoneally injected every day either FK506 at a dose of 1mg/kg/day (FK506 treated group) or physiological saline at 10ml/kg/day (Control group). Pr
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ograf, a solution containing 5mg FK506/ml, was diluted in physiological saline to obtain a final concentration. bone marrow cells were collected by flushing femoral shafts using sterile needles. Cells plated in 24-well plates were cultured for 6 days in α-minimal essential medium supplemented with 10% fetal bovine serum, 100U/ml penicillin, and 100 μg/ml streptomycin, 30 ng/ml macrophage-colony stimulating factor, and 100 ng/ml soluble Receptor Activator of NFκB Ligand. TRAP assay : Staining for tartrate-resistant acid phosphatase(TRAP) activity, a leukocyte acid phosphatase assay kit was used. At the end of the culture period, TRAP positive cells exhibiting ≧3 nuclei were counted as mature osteoclasts and TRAP positive cells with <3 nuclei were considered osteoclast precursors. Resorption pit assay : Osteoclasts were seeded onto dentine slices placed in each well of 24-well plates, and after the cells were scraped off from the dentine slices, excavated pits were stained with acid hematoxylin Photographs were taken under a light microscope at ×40 magnification, and total areas of resorption pits were analyzed by the computer soft of NIH Image. Less
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