Development of an osteoporosis model pro-culture by immunity restraint agent and molecular biology analysis of bone absorption mechanism

血液和尿液分析：虽然两组的血钙水平基本保持在正常范围内，但FK506治疗组的尿钙水平从给药第1周开始显著升高。FK506治疗组在给药第1周血骨钙素水平显著高于对照组，治疗后差异无统计学意义。在整个给药期间，FK506治疗组尿PYD和Dpd的总和显著高于对照组。在各种骨吸收因子中，FK506治疗组在给药第3周及之后的血PTH水平显著高于对照组。细胞培养：适应1周后，随机分为两组，每天腹腔注射FK506 1mg/kg/d （FK506处理组）或生理盐水10ml/kg/d（对照组）。将含有5mg FK506/ml的溶液在生理盐水中稀释得到终浓度。用无菌针冲洗股轴收集骨髓细胞。24孔板细胞在α-微量必需培养基中培养6天，α-微量必需培养基中添加10%胎牛血清、100U/ml青霉素、100 μg/ml链霉素、30 ng/ml巨噬细胞集落刺激因子、100 ng/ml可溶性NFκB配体受体激活剂。TRAP检测：使用白细胞酸性磷酸酶检测试剂盒检测抗酒石酸酸性磷酸酶（TRAP）活性。培养结束时，≥3个核的TRAP阳性细胞为成熟破骨细胞，≥3个核的TRAP阳性细胞为破骨细胞前体。吸收坑试验：将破骨细胞植入24孔板每孔放置的牙本质切片上，刮去牙本质切片上的细胞后，用酸苏木精染色挖掘出的坑，在光镜下×40放大拍照，利用NIH Image的计算机软件分析吸收坑的总面积。少

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 … More 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