Expression And Importance Of Core-binding Factor A1 In Fibroblast Exposed To Fluoride

The skeletal fluorosis includes bone sclerosis, osteomalacia, bone osteoporosis and extraperiosteal ossification. The soft tissue calcification and ossification plays an important role in the development of severe disabled skeletal fluorosis. Fibroblast (FB) , which is the main type of cell exists in the extraperiosteal soft tissue, belongs to the category of inducible osteogenic precursor cell (IOPC) because of its osteogenesis function under the non-physiological conditions. Our study showed that fluoride can induce expression of osteogenic phenotype (such as cbfa1, OCN, etc.) and enhance the potent osteogenesis function (the increase of Col I and osteogenesis nodules) of fibroblast. This study will pay attention on the expression of cba1, OCN, Col I in three-dimension culture system for providing the important clue to the mechanism of extraperiosteal ossification. At the same time, we try to find out the relationship between BMP-2 and cbfa1 in fibroblast treated with fluoride.The fibroblast cell line (L929) were cultured in three-dimension culture system (FPCL). The expression of cbfa1 and BMP-2 from mRNA to protein and the protein content of OCN and Col I in different time and fluoride-treated concentration in fibroblast were observed by RT-PCR, ELISA and IHC methods. The relationship between expression cbfa1 and BMP-2 in different time was discussed.1. Effects of fluoride on the level of cbfa1, BMP-2, OCN and Col I(1) The higher expression of cbfa1 mRNA was fond in group of 20 mg/L, the cbfa1 protein content were increased by fluoride in groups of 0.0001,0.001,10 and 20 mg/L at 2 h, 10 and 20 mg/L at 48 h, 10 mg/L at 72 h.(2) The increasing tendency of BMP-2 mRNA was observed in every fluoride groups, the protein content of BMP-2 were enhanced obviously in groups of 10 mg/L at 48 h and 1 mg/L at 96 h.(3) OCN protein content was increased in the group of higher fluoride-concentration (20 and/or 10 mg/L at 48,72 and 96 h.). (4)Col I protein content increased by 0.1,1,10 and 20 mg/L F- at 2,4 h.2. The same changing tendency was showed in fibroblast treated with fluoride in two-dimension and culture and FPCL(1) cbfa1: The level of cbfa1 mRNA and protein increased by fluoride both in two and three-dimension culture : The cbfa1 protein content increased in most fluoride groups in two-dimension culture, but only increased in some groups at 2, 48 and 72 h in three-dimension culture; The expression of cbfa1 mRNA was increased by 10 mg/L fluoride (P<0.05) in two-dimension culture, but little higher level only was fond in group of 20 mg/L in three-dimension.(2) BMP-2: The same changing tendency of BMP-2 mRNA and protein were observed in two of culture systems.(3)OCN: The effect of fluoride on the expression of OCN in two–dimension culture fibroblast was stronger than it was in three-dimension culture. (4) Col I: It had the same increased quantity in both two culture system.3. Both cbfa1 and BMP-2 were increased in fluoride-treated fibroblasts but no time relations were observed. We infer that BMP-2 may increase the level cbfa1 by stimulating other mediate factors.The study brought us some new ideas:1. The higher expression of cbfa1 and BMP-2 from mRNA and the protein content of OCN and Col I were enhanced by fluoride in fibroblast in FPCL. These results suggested that effects of fluoride on the expression of osteogenic phenotype and osteogenesis function in fibroblast.2. With some differences in degrees, the expression of cbfa1, BMP-2, OCN and Col I in fluoride-treated fibroblasts cultured in the two kinds of culture systems had the same changing tendencies. These results confirmed the effects of fluoride on fibroblast,on the other hand,they suggested that the two-dimension culture method should be the first selecting in study, and the three-dimension culture may be used for making the deep research. Ideal results will be acquired by uniting two-dimension culture with three- dimension culture.