| As one of the first factors have been found, Epidermal growth factor (EGF) may be important in regulating the proliferation,differentiation of epithelial cells. Determination of the molecules that may be required for eruption began with the isolation of epidermal growth factor (EGF) by Cohen and his discovery that its injection into rodents accelerated incisor eruption. So EGF may play a part in initiating eruption, but its real effection hasn't been found.The dental follicle (DF) and alveolar bone resorption are required for tooth eruption to occur, and the influx of mononuclear cells into dental follicle is critical for eruption. Prior to the onset of eruption, there is an influx of mononuclear cells into the coronal portion of dental follicle, with a concurrent increase in the numbers of osteoclasts that resorb alveolar bone to form an eruption pathway. Monocyte chemotactic protein-one (MCP-1) is prime candidate for recruiting the osteoclast precursors (mononuclear cells) into the DF. Because the peak influx of mononuclear cells is at day 3 post-natally in the rat, the positive correlation of this cellular influx with the peak gene expression for MCP-1 at day 3 post-natally is striking.The purpose of this present study was to evaluate the effects of of EGF on the secretion of MCP-1 in rat dental follicle cells, and the research was contributed to elucidate the mechanism of tooth eruption and provide theory for the orthodontic therapy of impacted teeth and tissue regeneration of tooth organs.Methods: 1. Cell culture: Developing mandibular first molar germs from 4～6day-old Wistar rat were isolated under stereomicroscope. Primary dental follicle cells were obtained by the methods of trypsin digestion and tissue culture, subsequently; dental follicle cells were cultured and subcultured. Cell origin was identified by the expression of Vimentin and Cytokeratin. The cells of the fourth passage were used.2. The effects of EGF on the proliferation of cells: 2×10~4cells were plated into 96 well microplates. After 24h inα-MEM containing 15% fetal bovine serum, the cells in each well were rinsed thrice inα-MEM. Controls were then cultured inα-MEM containing 0.5% serum, experimental cultures were grown in this plus different concentrations (0.5, 1, 5, 10, 50ng/ml) of epidermal growth factor in medium for 5 days. MTT assays were used to evaluate the proliferation of the control and experimental groups.3. The effects of EGF on the expression of MCP-1 in dental follicle cells: Cells were plated into each 70mm dia culture dish at the concentration of 1×10~5 inα-MEM containing 0.5% serum for 5 days, and then the cells were incubated with 10ng/ml EGF for different hours (0, 0.5, 1, 3, 6). Subsequently, the total RNA was then extracted from each sample for Reverse transcription-polymerase chain reaction analysis. The results were analyzed by SPSS13.0 software package.Results:1. The typical characterization of cultured cells was plemorphism and there were two major cell types: some were cuboidal or polygonal; some were elongated, spindle-shaped, the results of immunocytochemistry indicated that dental follicle cells expressed Vimentin positively while the expression of Cytokeratin was negative, which indicated that the cells were originated from mesenchyme.2. Epidermal growth factor at the concentration of 5～10ng/ml significantly stimulated the proliferation of the dental follicle cells (P<0.05), and the prime effective concentration of EGF on dental follicle cells was 10ng/ml. 3. Epidermal growth factor had a dramatic effect on MCP-1 expression, resulting in peak expression at 3 hr after incubation (P<0.01) with a decline at later times.Conclusion:Proper concentrations of epidermal growth factor promote dental follicle cellproliferation, and it enhances MCP-1 transcription for the ultimate production ofMCP-1 for mononuclear cells recruitment in vitro. |
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