| Objective: To investigate the regulatory ability of Osteoking on adipogenic differentiation of rat BMSCs and the action mechanism in osteoporosis prevention and treatment.Methods: Via whole bone marrow adherence method,BMSCs were isolated from femurs and tibias of juvenile SD rats;immunohistochemical staining was to measure the purity of BMSCs;Osteoking was diluted by 500 times,1000 times,2000 times and 4000 times,and respectively divided into 2×10-3 group,1×10-3 group,0.5×10-3 group,0.25×10-3 group and control group(solvent);MTT method was to detect the cytotoxicity of Osteoking in various concentrations against BMSCs;BMSCs initiated adipogenic differention with the intervention of Osteoking in various concentrations;On the 21 d after Osteoking intervention,the adipogenic differention status was observed by microscope with Oil Red O staining and quantitatively measured by microplate reader;On the 7d,14 d,21d after Osteoking intervention,the relative mRNA expressions of peroxisome proliferator activated receptor(PPAR)γ and CCAAT/ enhancer binding protein(C/EBP)α were quantitatively detected by Realtime-PCR.Results: ? Rat BMSCs(neonatal rat)were obtained via cell adherence method;? By fluorescence inversion microscope,immunohistochemical staining displayed CD44 positive(95.47%)and CD19(-)in BMSCs,which indicated the successful culture of BMSCs with high purity;? MTT method indicated no statistical significance in BMSCs between Osteoking intervention groups(0.5d,1d,4d,21d)and control group(P>0.05).It indicated that Osteoking had no toxic effect on BMSCs proliferation and cell viability regardless of early or late stages;? On the 21 d after intervention,microscopic observation indicated Osteoking of various concentrations had inhibitory effect on adipogenic differentiation of BMSCs after Oil Red O staining,which was concentration-dependent(higher inhibition by higher concentration).The OD values from microplate reader were 1.104±0.414,0.785±0.085,0.836±0.138,1.008±0.369 and 1.000±0.291.The differences between 2×10-3 group,1×10-3 group and control group were statistically significant(P<0.05),which were in accordance with staining results;(5)Osteoking inhibited the generation of adipocytes and blocked the expression of adipocyte-specific transcription factors.On the 7d,14 d and 21 d after intervention,results of Realtime-PCR indicated down-regulated gene expressions of PPARγ and C/EBPα comparing to control group,and the difference was statistically significant(P < 0.05).Conclusion: Osteoking inhibited adipogenic differentiation of rat BMSCs via down-regulation the expressions of PPARγ and C/EBPα,which was other functional mechanism of Osteoking in the prevention and treatment of osteoporosis. |
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