Effect Of TRAF6 Gene Silencing On Proliferation And Osteoblast Differentiation Of HPDLCs With LPS Challenge

Objective:Tumor necrosis factor receptor related factor 6(TRAF6)in human periodontal ligament cells(HPDLCs)was silenced by Small interfering RNA(siRNA).The purpose of this study is to detect the change on proliferation and osteoblast differentiation of TRAF 6 gene silencing cells under LPS challenge.Methods:(1)HPDLCs were isolated from periodontal ligament with method which based on combination of tissue block and mixed enzyme digestion.The immunocytochemistry was used to disclose the expression of cytokeratin and vimentin.(2)The experiment was divided into four groups: TRAF6 siRNA group,Control SiRNA group,transfection reagent group,blank control group.Lipofectamine method was used to transfected TRAF6 siRNA and control siRNA into HPDLCs,transfection reagent group only added the same amount of transfection reagent,blank control group without any disposal.And then the cells were stimulated by LPS.RT-PCR and Western blot were carried out for testing the expression of TRAF6.(3)The proliferation of TRAF6 knockdown cells after LPS challenge of 24 h or 48 h were tested by Cell Counting Kit-8(CCK-8).(4)After transfection,cells were cultured with LPS(10?g/ml)and osteogenic differentiation induction medium for 3 days.Alkaline phosphatase assay kit was used to detect the activity of ALP,the expressions of Runx-2 and type I collagen(Col-I)gene were detected by RT-PCR.Results:(1)Immunocytochemistry showed the cells were HPDLCs.(2)Compared with other three groups,The expression of TRAF6 in TRAF6 siRNA group was significantly decreased(p<0.001).(3)After stimulation with LPS(10?g / ml)for 24 and 48 hours,the a value of TRAF6 siRNA group was significantly lower than that of other two groups(p<0.001).(4)After 3 days of osteogenic induction culture,the expression of ALP in TRAF6 siRNA group was significantly higher than the other three groups(p<0.05).The expression of Runx-2 and Col-I mRNA in TRAF6 siRNA group was also higher than that in the other three groups(p<0.05).Conclusion:TRAF6 gene silencing can inhibit the proliferation of HPDLCs and stimulate the osteogenesis of HPDLCs in LPS stimulation.It is speculated that TRAF6 is involved in the development of periodontal disease and is a potential therapeutic target for periodontal disease.