RESUMO
Objective @# To investigate the effect of miR-214 on the osteogenic differentiation of dental follicle cells (DFCs).@*Methods@#Purified DFCs were cultured in vitro by bidirectional differential passage, with the untransfected DFCs as the control group (DFCs group). The expression of miR-214-3p in DFCs was upregulated and downregulated by transfection of miR-214-3p(miR-214 mimics group) or miR-214-3p inhibitors(miR-214 inhibitor group) into DFCs. The expression levels of miR-214, alkaline phosphatase (ALP), osteonectin (OSN) and runt-related transcription factor-2(RUNX-2) were detected by qRT-PCR after 7 days of osteogenesis induction, the protein expression levels of RUNX-2 and β-catenin were detected by western blot, and the formation of mineralized nodules was observed with alizarin red staining after 14 days of osteogenesis induction. @*Results @# Compared with the DFCs group, in the miR-214 mimics group, the expression of miR-214 was upregulated after 7 days of osteogenesis induction. The mRNA expression of ALP, OSN and RUNX-2 in the miR-214 mimics group was lower than that in the DFCs group, but only ALP in the two groups was statistically significant (P > 0.05); the mRNA expression of ALP, OSN and RUNX-2 in the miR-214 inhibitor group was higher than that in the DFCs group, and the difference was statistically significant (P < 0.05). The protein expression of RUNX-2 and β-catenin in the miR-214 mimics group was lower than that in the miR-214 inhibitor group. The number of calcified nodules in the miR-214 mimics group was significantly less than that in the DFCs group, while that in the miR-214 inhibitor group was significantly higher than that in the DFCs group. @*Conclusion@#The upregulation of miR-214 can downregulate the expression of β-catenin, can inhibit the expression of ALP, OSN and RUNX-2 related to osteogenesis, and can inhibit osteogenic differentiation. The downregulation of miR-214 demonstrated the opposite results; miR-214 may downregulate the expression of β-catenin and inhibit the osteogenic differentiation of DFCs.
RESUMO
Objective:To develop a simple culture and purifying method for rat dental follicle cells.Methods:The upper and lower first and second intact molar germs of SD rat were separated. Then, dental follicle and enamel organ were stripped together, minced into little pieces, digested with collagenase and cultured. Dental follicular cells were purified by differential passage and indentified by immunohistochemical staining of vimentin and cytokeratin. Results:The primary cells were mixed, consisting of dental follicle cells and enamel organ cells. After differential passage, the cells of fourth passage became purified dental follicle cells. Purified dental follicle cells were elongated spindle or triangle in shape, positive for vimentin and negative for cytokeratin.Conclusion:Dental follicle cells can be purified by several differential passages from the mixed primarily cultured cells.