| Objective: To study the apoptosis effect of different concentrations of DEX impact on osteoblast at different times and to investigate the possible mechanism in glucocorticoid-induced osteoporosis.Methods: Rat osteoblasts were isolated from skull of newborn SD rats by enzymatic digestion methods.The isolated and cultured rat osteoblasts were observed by inverted phase contrast microscope and transmission electron microscope cell morphology,and identified by alkaline phosphatas e BCIP/NBT staining and Alizarin red mineralized nodules staining.The third generation cells were tested by 5*10-5M,5*10-6M,5*10-7M,5*10-8MDEX for 12 h,24 h,48 h.The survival rate of osteoblast after intervention by different concentrations of DEX at different times was detected by cck8 method.The apoptosis rate of osteoblasts was detected by flow cytometry,real-time fluorescence quantitative PCR was used to detect the expression of alkaline phosphatase、type I collagen 、caspase-3 and caspase-9 after intervention by different concentrations of DEX at different times.Westernblotting was used to detect the expression of caspase-3 protein in osteoblasts.Results: 1.The cells were adherent growth,showed spindle,triangle,polygon,having two or three protrusions and the cells were stained positive by alkaline phosphatase staining and Alizarin red staining.2、The cck8 results showed that,compared with the control group,the inhibitory effect of DEX on the proliferation of osteoblasts was gradually enhanced,and the inhibition of 5*10-5M was the strongest.It suggested that DEX inhibited the proliferation of osteoblast in a concentration dependent manner.3.Annexin-FITC / PI flow cytometry results showed that 5*10-5M,5*10-6M,5*10-7M,5*10-8M DEX promoted the apoptosis of osteoblasts,and the apoptosis effect of DEX on osteoblast increased with the extension of drug concentration and time.4.PCR results showed that different concentrations of DEX can cause the decrease expression of alkaline phosphatase and type I collagen,and increase expression of caspase-3 and caspase-9,showing concentration dependence and time dependence.5.Results of western-blotting detection showed that the expression of caspase-3 protein was enhanced after 24 hours of DEX intervention,and the expression of caspase-3was showed concentrationdependence.Conclusion: DEX may participate in the occurrence of Glucocorticoid-induced Osteoporosis by inducing apoptosis of osteoblast and then inhibiting bone formation. |
