Effect Of The Expression Of CTGF In Fibroblast Induced By Fluoride

Connective tissue growth factor ( CTGF ) can promote proliferation and differentiation of chondrocyte and osteoblast ( OB ) . It is of great importance in bone development and bone mass maintenance . Investigations have proved that CTGF can also stimulate cell proliferation and collogen secretion of fibroblast . TGF-βhas the same usage with CTGF in biological research and it is also the factor which stimulate the expression of CTGF effectively . We observe the characteristics of FB cultured in vitro and protein expression , we attempt to analyze the interrelation between TGF-βand CTGF in the effect of fluorid. By doing that we can provide an important evidence to explain the cause of the non-physiological osteogenesis leading to extraperiosteal ossification .Fluoride can stimulate the enhancement of osteogenic phenotype and osteogenic function. Based on inferences above , we investigated the effect of fluorid on FB line of mouse ( L929 ) and mouse's osteoplastic cell of skull ,we may observe on FB CTGF mRNA and protein expression affected by fluoride in the 2-dimensional ( 2D ) and 3-dimensional ( 3D ) cultured system . We investigate simultaneously the expression of TGF-βprotein in FB following the treatment with fluoride . We expect the relationship between TGF-βand CTGF , which produce the effect of the expression in FB exposed to fluoride . We investigated the effects and importance of fluoride on FB .1,The expression of CTGF mRNA and protein in fluoride-treated FB(1) By using the method of Elisa , we detected the protein level of CTGF in supernatant fluid in fluoride-treated FB . Compared with the group of 0 mg F-/L , either in 2D or 3D cultured system , the content of CTGF protein was increased obviously in the group of higher fluoride dosage and later stimulation of fluoride .(2) By using the method of IHC , we detected the expression of the masculine protein in cytoplasm of FB , as a result , we found that fluoride can stimulate the masculine protein of CTGF . Compared with the CTGF protein expression in the group of 0 mg F-/L , it in the group of fluoride-treated FB in 96h enhanced apparently in 3D cultured system .(3) By using the method of RT-PCR , we detected the expression of the fluoride-treated FB CTGF mRNA in 48h . The result showed that the expression of CTGF mRNA had been enhanced in the group of higher fluoride dosage in 2D and 3D cultured system . It , however , did not rich the apparent degree in statistics .Above-mentioned result shows that the expression of CTGF in FB has been stimulated by fluoride in 2D and 3D cultured system . CTGF stimulated the function of osteogenesis and it may have an important usage in osteoplastic potency of FB .2,Effect of the expression of TGF-βin fluoride-treated FB and OB(1) We used the method of Elisa to check TGF-βprotein level in supernatant fluid in fluoride-treated FB . The result shows that there is a different tendency in the expression of TGF-βprotein between the 2D and 3D cultured-FB . The expression of TGF-βprotein was decreased or reduced obviously in 2D culture . In 3D culture , compared with 0 mg F-/L the expression of TGF-βprotein in group of 0.0001 mg F-/L was more increased obviously than in other group .(2) By using the method of Elisa , we detected the protein level of TGF-βin supernatant fluid in fluoride-treated OB . According to the experiment , the protein level affected by fluoride was not changeable obviously in protophase , was fortified in intermediate stage and was depressed obviously in last period .Above-mentioned result shows that in two cultured systems reaction of FB TGF-βprotein expression affected by fluoride differs a little . It reminds us to use different cultured methods in the future research so that we can gain allround results .3,The expression of TGF-βand CTGF in FB affected by fluoride and the interrelation between themWe did not find obvious connection between the expressions of FB TGF-βand CTGF in the experiment . This is probably because two factors work respectively or because of limitation of the experimental condition . It is necessary for us to make a further research .4,The expression of CTGF mRNA in OB affected by fluoride is similar to FB affected by fluoride , but different from expression of proteinThe expressive tread of CTGF mRNA in FB and OB is extremely similar and it is in agreement with the expression of bone growth factor mRNA which we have research before . The fluoride has little effect on the expression of CTGF protein in OB ,we still need a further research .We , in the project , will find the effect of the expression of CTGF on different cultured condition induced by fluoride , and contrast the expression of CTGF in fluoride-treated FB with that in OB under the same condition . The studies above will demonstrate that the fluoride could stimulate the expression of CTGF in FB with two cultured condition . The expressive tread of CTGF in FB with 2D culture and OB is extremely similar . The interrelationship of TGF-βand CTGF in fluoride-treated FB is not coincident with our hypothesis . Several improvement need to be put forward in our further study . CTGF may be an important factor in the project which fluoride promotes osteogenesis of FB . It can give clues to thestudies of other similar diseases .