Objective To investigate the expression and the binding characteristics of leptin receptor (Ob-R) on human osteoblast. Methods The human osteoblasts were cultured and identified by cell biological criteria. 125I-lepti...Objective To investigate the expression and the binding characteristics of leptin receptor (Ob-R) on human osteoblast. Methods The human osteoblasts were cultured and identified by cell biological criteria. 125I-leptin was prepared by chloramine-T method and purified by PAGE. Radioligand binding assay of receptor and Scatchard plot were performed to identify the binding properties of the osteoblasts. Results Morphologically, the cultured cells showed analogical phenotype to osteoblast, and were positive in histochemical and immuno-fluorence staining for alkaline phosphatase and osteocalcin, respectively. The Alizarin Reds staining showed that mineralization was also developed by supplementation with ascorbate and β-glycerophosphate. The percentages of 125I-leptin binding to osteoblast were 16.7%±2.3% and 6.1%±1.4% without and with unlabelled leptin, respectively. From the saturation assay and by Scatchard plotting, a single class of high-affinity binding sites of Ob-R for leptin was identified with an apparent kd of 0.11±0.06 nmol/L and a Bmax of 1.7±0 3 nmol/10 5 cells. Conclusion The human osteoblasts are target of leptin, with expression of Ob-R. The leptin could possibly regulate the growth, prolifiration, function of osteoblasts and even bone remoldeling.展开更多
文摘Objective To investigate the expression and the binding characteristics of leptin receptor (Ob-R) on human osteoblast. Methods The human osteoblasts were cultured and identified by cell biological criteria. 125I-leptin was prepared by chloramine-T method and purified by PAGE. Radioligand binding assay of receptor and Scatchard plot were performed to identify the binding properties of the osteoblasts. Results Morphologically, the cultured cells showed analogical phenotype to osteoblast, and were positive in histochemical and immuno-fluorence staining for alkaline phosphatase and osteocalcin, respectively. The Alizarin Reds staining showed that mineralization was also developed by supplementation with ascorbate and β-glycerophosphate. The percentages of 125I-leptin binding to osteoblast were 16.7%±2.3% and 6.1%±1.4% without and with unlabelled leptin, respectively. From the saturation assay and by Scatchard plotting, a single class of high-affinity binding sites of Ob-R for leptin was identified with an apparent kd of 0.11±0.06 nmol/L and a Bmax of 1.7±0 3 nmol/10 5 cells. Conclusion The human osteoblasts are target of leptin, with expression of Ob-R. The leptin could possibly regulate the growth, prolifiration, function of osteoblasts and even bone remoldeling.
