Effects Of Exogenous Calcium On Proliferation And Wnt Signaling Pathway Of Mouse Osteoblasts Exposed To Fluoride

[Objective] To investigate the underlying mechanism of fluoride-induced osteoblasts proliferation and appropriate dose of calcium antagonize to fluorosis, we evaluated the expression of related genes and proteins on Wnt signaling pathways in fluorosis osteoblasts respectively treated with inhibitor of Wnt pathway and different dose of calcium, which provides the theoretical basis to preventing fluorosis.[Methods] Osteoblasts were collected from calvarium of new born mouses by enzyme digestion, and determined by HE, Giemsa, AKP, and calcification nodule staining. The third generation of osteoblasts was divided into 7 groups randomly. Each group treated as follows:Control group was cultured with normal DMEM complete medium; Fluoride group was added 10-8,10-7,10-6,10-5, 10mol/L concentration of fluoride to DMEM complete medium; Fluoride and inhibitor group was added 10-6mol/L concentration of fluoride and 0.5μg/mL DKK1 to DMEM complete medium; Inhibitor group was only added 0.5μg/mL DKK1 to DMEM complete medium; Fluoride and CaⅠ group was added 1mmol/L concentration of calcium to fluoride group’s culture; Fluoride and CaⅡ group was added 2mmol/L concentration of calcium to fluoride group’s culture; Fluoride and CaⅢ group was added 4mmol/L concentration of calcium to fluoride group’s culture. The time of experiment cycle is 24h. The proliferation of osteoblasts were detected by MTT, and the expression on Wnt pathways related genes and proteins were detected by real-time PCR, immune fluorescence, Western blotting, and laser scanning confocal technologies.[Result] 1. Osteoblasts were isolated from calvarium of new born mouses by enzyme digestion, and identified by HE, Giemsa, AKP, and calcification nodule staining successfully.2.10-8,10-7, 10-6 mol/L concentration of fluoride stimulated the proliferation of mouse osteoblasts, 10-5mol/L concentration of fluoride stimulated the proliferation of mouse osteoblasts unobtrusively, 10-4mol/L concentration of fluoride inhibited the proliferation of mouse osteoblasts.3. The increment rate of the mouse osteoblasts induced by fluoride is the highest when cultured for 24h in vitro, compared with 48h and 72h.4. The inhibitor of Wnt signaling pathway obviously inhibited the proliferation of mouse osteoblasts induced by fluoride5.10-6mol/L concentration of fluoride promoted the expression of Wnt3a, P-Catenin, C-myc gene in mouse osteoblasts, adding 2mmol/L concentration of calcium effectively inhibited the expressions of proliferation related gene in mouse osteoblasts.6.10-6mol/L concentration of fluoride promoted the expression of β-Catenin, Wnt3a, C-myc and CyclinDl protein in mouse osteoblasts, simultaneously inhibited the expression of GSK3β protein, adding 2mmol/L concentration of calcium effectively inhibited the expressions of proliferation related protein in mouse osteoblasts.[Conclusion] 10-6mol/L concentration of fluoride significantly promoted the proliferation of mouse osteoblasts by activating the Wnt signaling pathways, supplementing 2mmol/L concentration of calcium antagonized the toxic effects induced by fluorosis.