成骨细胞对葡萄糖水平的感知作用

背景：新近研究表明，骨钙素可提高胰岛素分泌水平、胰岛素敏感性，并且可阻止脂肪的积累，参与糖脂代谢，发挥此作用的主要是羧化不全骨钙素。 目的：观察不同浓度葡萄糖对成骨细胞羧化不全骨钙素分泌水平的影响。 方法：切下人肋骨骨小梁剪碎，胰酶消化后以PBS彻底冲洗直至于倒置显微镜下观察骨片表面及清洗液中无粘着及漂浮的细胞，以DMEM完全培养液清洗骨小梁1次，移入培养瓶内培养,每周更换培养液1次，1周后可见成骨细胞自骨片移出，4~6周细胞融合成单层可作传代培养。取生长活跃的传二三代细胞接种于6孔板中分成5组，待细胞融合至80%以含5.6，7.6，9.6，12.6，20.6 mmol/L葡萄糖的培养基刺激成骨细胞，同时加入维生素K2使其在培养液中最终浓度为10-5 mol/L。培养半小时后收集上清液，以放免法检测成骨细胞培养上清液中羧化不全骨钙素水平，并以上清液中总骨钙素进行校正，计算羧化不全骨钙素率。 结果与结论：在不同葡萄糖浓度下成骨细胞羧化不全骨钙素分泌水平不同，7.6，9.6，20.6 mmol/L葡萄糖浓度组羧化不全骨钙素率高于5.6 mmol/L葡萄糖浓度组(0.27±0.02)，(0.29±0.04)，(0.12±0.02)%，P < 0.05]。提示成骨细胞可感知葡萄糖浓度变化,在5.6~9.6 mmol/L葡萄糖刺激下成骨细胞可呈剂量依赖性地提高自身羧化不全骨钙素分泌水平，进一步提高葡萄糖浓度时羧化不全骨钙素水平反而降低。

Role of osteoblasts in the sense of glucose

BACKGROUND: Recent study showed that osteocalcin may elevate Insulin secretion and sensitivity, prevent the fat accumulation, play a role in the metablism of glucose and lipid. Undercarboxylated osteocalcin works as the main role. OBJECTIVE: To investigate the effect of different concentrations of glucose on osteoblast undercarboxylated osteocalcin. METHODS: The rib trabeculae were resected and broken, trypsinizated and washed completely by PBS. Bone surface and non-adhesive floating cells in cleaning fluid were observed with inverted microscope. Rib trabeculae was washed by DMEM culture medium once, and cultured in culture bottle. The culture liquid was replaced by new one once a week. The osteoblast was moved from the sclerite a week later. The cells were fused monolayer and could be subcultured 4 to 6 weeks later. The active second or third generation cells were inoculated to 6-pore plate forming 5 groups. Osteoblast were stimulated by 5.6 mmol/L, 7.6 mmol/L, 9.6 mmol/L, 12.6 mmol/L, 20.6 mmol/L glucose medium respectively after the 80% cells were fused, the vitamin K2 was added into the culture liquid until the concentration of it to be 10-5 mol/L. Supernatant was collected after half hour culturing, the undercarboxylated osteocalcin level were detected with RIA test kit, and corrected it as the total the undercarboxylated osteocalcin, calculated the carboxylated incomplete osteocalcin rate. RESULTS AND CONCLUSION: The rate of osteoblast carboxylated incomplete osteocalcin was different under different concentration glucose. The rate of 7.6 mmol/L, 9.6 mmol/L, 20.6 mmol/L concentration glucose groups were higher than that of 5.6 mmol/L glucose group (0.27±0.02)%, (0.29±0.04)%, (0.12±0.02)%, P < 0.05]. It is indicated that osteoblast could sense the change of glucose concentration by regulating the secretion of the undercarboxylated osteocalcin between the concentration of 5.6mmol/L to 9.6mmol/L, while the carboxylated incomplete osteocalcin decreased as the concentration of glucose increased.