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Objective: The study was designed to investigate the molecular mechanism of quercitrin on osteogenic differentiation and adipogenic differentiation of rBMSCs. Methods: rBMSCs were harvested from SD rats, and determination of alkaline phosphatase (ALP) activity, quantification of mineralization by Alizarin Red S staining, and the mRNA expression of osteogenic differentiation markers (Runx2, BMP-2, and OSX) by RT-PCR after rBMSCs stimulated by osteogenic induction with (0.1–10) µg/mL of quercitrin, quantification of Lipid droplet by Oil Red O staining and the mRNA expression of adipogenic differentiation marker (PPARγ C/EBPα and aP2) by RT-PCR after rBMSCs stimulated by adipogenic induction with (0.1-10) µg/mL of quercitrin. Results: Quercitrin can up-regulate the mRNA expression of osteogenic differentiation markers (Runx2, BMP-2, and OSX) and increase ALP activity and mineralization after osteogenic induction, on the other hand quercitrin can suppress the mRNA expression of adipogenic differentiation markers (PPARγ C/EBPα and aP2) and decrease lipid droplet after adipogenic induction. Conclusion: This study suggested that quercitrin not only stimulated osteogenic differentiation but also inhibited adipogenic differentiation of rBMSCs, which was associated with the up-regulation of Runx2, BMP-2, and OSX mRNA expression and the down-regulation of PPARγ C/EBPα and aP2 mRNA expression.
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ABSTRACT:Objective To investigate the effects of phenytoin (PHT)on the secretion of vascular endothelial growth factor (VEGF)and stem cell factor (SCF)based on the establishment of indirect co-culture system of rat bone marrow mesenchymal stem cells (BMSCs)and vascular endothelial cells (VECs).Methods Indirect co-culture model of rat BMSCs and VECs was established.Experimental groups:indirect co-culture groups (PHT concentrations were 0,20 and 40 μg/mL);the control group:BMSCs culture group and VECs culture group (PHT concentrations were 0,20 and 40μg/mL).The contents of VEGF and SCF in the culture supernatant were measured using double antibody sandwich ABC-ELISA method on cultivation days 2,4,6.Results ELISA assay of the rBMSCs and rVECs in indirect co-culture supernatants,collected on culture days 2,4 and 6 showed that:① VEGF:On culture day 2,VEGF level in the co-culture groups was significantly higher than those in BMSCs group (P SCF increased as the incubation time increased;but as the incubation time increased, PHT concentration of 40 μg/mL made SCF content decrease.Each group did not significantly differ (P > 0.05 ).Conclusion PHT promotes the secretion of VEGF and may reduce the secretion of SCF.
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Objective:To investigate the effects of PHT on the differentiation of rat bone marrow mesenchymal stem cells(BMSCs) into vascular endothelial cells(VECs).Methods:rBMSCs were cultured in indirect coculture system with VECs and independent culture system in the presence of phenytoin at 0,20 and 40 μg/ml respectively.After 1 4 d culture the mRNA expression levels of the intercellular adhesion molecule-1 (ICAM-1 ),vascular cell adhesion molecules-1 (VCAM-1 ),vascular endothelial growth factor re-ceptor -2(KDR)and Runt-related transcription factor 2 (RUNX2)were detected by real-time PCR.Results:The expression levels of ICAM-1 ,KDR and RUNX2 were upregulated in rBMSCs by PHT treatment.The expression levels of ICAM-1 ,KDR and RUNX2 in the co-culture groups were higher than those in the independent culture groups of rBMSCs treated by PHT at the same concentra-tion.The expression level of VCAM-1 in co-culture groups was lower than that in the independent culture groups.Conclusion:PHT may promote rBMSCs differentiation into rVECs in the co-culture system.
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Objective To investigate the effect from mechanical stimulation and osteogenic chemical inductor on osteoblastic differentiation markers and formation of calcified nodules in rat bone mesenchymal stem cells (rBMSCs). Method The rBMSC were cultured in medium contained with or without osteogenic chemical inductor. The cyclic biaxial mechanical strain (2%), at a frequency of 1 Hz, was applied to the rBMSCs for periods of 2 hours each time, at intervals of 2 hours, 3 times every day, lasting 3 days and 6 days, respectively. The mRNA expression of alkaline phosphatase (ALP), collagen type I (COL I) and osteocalcin (OCN) were analyzed with real time fluorescent quantitation reverse transcription polymerase chain reaction(qRT-PCR) and formation of calcified nodules were detected with alizarin red staining method. Results The mRNA expression of ALP, COL I and OCN were significantly increased in induced group compared with that in the corresponding uninduced group and calcified nodule was observed in the osteogenic chemical inductor group after 6 days with mechanical stimulation. Conclusions Osteogenic chemical inductor and mechanical stimulation can promote the osteoblastic differentiation of rBMSCs.