A series of experimental methods including 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) test,alkaline phosphatase (ALP) activity measurement,Oil Red O stain and measurement,mineralized functi...A series of experimental methods including 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) test,alkaline phosphatase (ALP) activity measurement,Oil Red O stain and measurement,mineralized function expression and quantitive real time RT-PCR (qRT-PCR) were employed to assess the effect of Nd3+ and Sm3+ on the proliferation,differentiation and mineralization function of primary osteoblasts (OBs) in vitro at cell and molecular levels.The experimental results suggest that concentration,culture time and ion species are pivotal factors for switching the biological effects of rare earth ions from toxicity to activity,from damage to protection,or from down-regulation to up-regulation.展开更多
Background Calcitonin gene-related peptide （CGRP）, a sensory neuropeptide, affects osteoblast proliferation and bone formation. However, the mechanisms are not fully understood. Monocyte chemoattractant protein-1 （...Background Calcitonin gene-related peptide （CGRP）, a sensory neuropeptide, affects osteoblast proliferation and bone formation. However, the mechanisms are not fully understood. Monocyte chemoattractant protein-1 （MCP-1） is a chemokine that stimulates the migration of monocytes and plays important roles in regulating bone remolding during fracture repair. In this study, we investigated the effects of CGRP on proliferation and MCP-1 expression in cultured rat osteoblasts. Methods Primary rat osteoblasts were isolated from fetal rats calvariae. Cells were exposed to gradient concentrations （10^-9 to 10^-7 mol/L） of CGRP. Protein and mRNA levels of MCP-1 were quantified by Western blotting and semiquantitative reverse transcdption-polymerase chain reaction, respectively. The protein level of MCP-1 was investigated and compared in cell culture media by enzyme linked immunosorbent assay （ELISA）. Phospho-extracellular signal-regulated kinase （ERK） expression was detected by Western blotting. Cell proliferative activity was measured by 3-（4,5-dimethylthiazol-2-yl）-2, 5-diphenyl tetrazolium bromide （MTT） and BrdU assay. The effects of MAPK/ERK kinase （MEK）-inhibitor U0126 on CGRP-induced MCP-1 expression in primary rat osteoblasts were examined. Results CGRP effectively enhanced primary rat osteoblast proliferation and led to significant increases in the expression of MCP-1 mRNA and protein in time- and dose-dependent manners. CGRP activated the ERK pathway. Pretreatment of cultured rat osteoblasts with MEK inhibitor U0126 resulted in dose-dependent inhibitions of CGRP-induced MCP-1 mRNA and protein levels. Thus, CGRP promoted cell proliferation and stimulated MCP-1 expression in cultured rat osteoblasts. Conclusion These studies document novel links between CGRP and MCP-1 and illuminate the effects of CGRP in regulating bone remodeling.展开更多
基金supported by the National Natural Science Foundation of China (20971034)the Natural Science Foundation of Hebei Province (B2009000161)+2 种基金Foundation for Key Program of Ministry of Education of China (208018)Returned Scholars of Hebei Province (207041)Natural Science Foundation of Hebei University
文摘A series of experimental methods including 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) test,alkaline phosphatase (ALP) activity measurement,Oil Red O stain and measurement,mineralized function expression and quantitive real time RT-PCR (qRT-PCR) were employed to assess the effect of Nd3+ and Sm3+ on the proliferation,differentiation and mineralization function of primary osteoblasts (OBs) in vitro at cell and molecular levels.The experimental results suggest that concentration,culture time and ion species are pivotal factors for switching the biological effects of rare earth ions from toxicity to activity,from damage to protection,or from down-regulation to up-regulation.
文摘Background Calcitonin gene-related peptide （CGRP）, a sensory neuropeptide, affects osteoblast proliferation and bone formation. However, the mechanisms are not fully understood. Monocyte chemoattractant protein-1 （MCP-1） is a chemokine that stimulates the migration of monocytes and plays important roles in regulating bone remolding during fracture repair. In this study, we investigated the effects of CGRP on proliferation and MCP-1 expression in cultured rat osteoblasts. Methods Primary rat osteoblasts were isolated from fetal rats calvariae. Cells were exposed to gradient concentrations （10^-9 to 10^-7 mol/L） of CGRP. Protein and mRNA levels of MCP-1 were quantified by Western blotting and semiquantitative reverse transcdption-polymerase chain reaction, respectively. The protein level of MCP-1 was investigated and compared in cell culture media by enzyme linked immunosorbent assay （ELISA）. Phospho-extracellular signal-regulated kinase （ERK） expression was detected by Western blotting. Cell proliferative activity was measured by 3-（4,5-dimethylthiazol-2-yl）-2, 5-diphenyl tetrazolium bromide （MTT） and BrdU assay. The effects of MAPK/ERK kinase （MEK）-inhibitor U0126 on CGRP-induced MCP-1 expression in primary rat osteoblasts were examined. Results CGRP effectively enhanced primary rat osteoblast proliferation and led to significant increases in the expression of MCP-1 mRNA and protein in time- and dose-dependent manners. CGRP activated the ERK pathway. Pretreatment of cultured rat osteoblasts with MEK inhibitor U0126 resulted in dose-dependent inhibitions of CGRP-induced MCP-1 mRNA and protein levels. Thus, CGRP promoted cell proliferation and stimulated MCP-1 expression in cultured rat osteoblasts. Conclusion These studies document novel links between CGRP and MCP-1 and illuminate the effects of CGRP in regulating bone remodeling.
