Research On Effect Of TNF-? On The Expression Of Ephrin/Eph In Periodontal Ligament Fibroblasts

With the development of various orthodontic techniques and methods in oral orthodontic treatment,the orthodontic program has been gradually optimized,but the long of orthodontic treatment and the discomfort in the process treatment are still the problems that the general orthodontic patients are facing.Orthodontic tooth movement depends on periodontal tissue remodeling with the action of orthodontic force,and bone remodeling is particularly important in this process.To shorten the time of orthodontic treatment and to accelerate the speed of orthodontic tooth movement have become one of the research hotspots in orthodontic treatment in recent years.Similarly,in the process of periodontitis,the loss of alveolar bone will lead to tooth loss,and how to slow down the damage of alveolar bone is also an urgent problem to be solved in the treatment of periodontitis.Humans periodontal fibroblasts(HPLF),located between alveolar bone and cementum.As the main components of human periodontal fibroblasts,they can secrete various cytokines to participate in the metabolism and remodeling of periodontal tissues.In recent years,it has been found that ephrin/Eph signaling pathways play an important role in the process of bone tissue remodeling,but there are few studies on the expression of ephrin/Eph in periodontal fibroblasts under the state of inflammation.TNF-? is one of the most important inflammatory factors.To know about ephrin/Eph expression under the action of the TNF-? in the periodontal ligament fibroblasts,and the relationship between ephrin/Eph pathways with other pathways could provide the reference for orthodontic treatment and the further study of alveolar bone reconstruction in the treatment of periodontitis.Objective:1.To detect the changes of the expressions of ephrinB2,EphB4 and EphA2 in periodontal membrane fibroblasts under different concentrations of TNF-? at different time.2.To detect the expressions of EphB4 in the gingival tissue of periodontitis patients.Methods:1.The periodontal tissue was extracted from normal teeth which needed to be removed due to orthodontic treatment.The growth status and shape characteristics were observed by tissue mass method.The periodontal fibroblasts were detected by IHC.2.The expressions of ephrinB2,EphB4 and EphA2 at mRNA level and protein levels of ephrinB2 and EphB4 were detected by RT-PCR and Western blot at 24 h and 48 h,respectively.3.The difference of expression of EphB4 in gingival tissue between healthy subjects and periodontitis was detected by IHC.Results:1.Primary human periodontal fibroblasts were successfully cultured,and the growth rate of periodontal fibroblasts was faster after passage.The results of immunohistochemistry showed that vimentin was positive and keratin was negative.2.The mRNA expression levels of ephrinB2 and EphB4 decreased after 24 h after TNF-? treatment(P<0.05).There was no significant difference in EphA2 mRNA expression between the two groups.After 48 h,0.1ng/ml TNF-? increased EphB4 mRNA expression(P<0.05),but had no effect on ephrinB2/EphA2 mRNA expression.Under 1ng/ml TNF-?,there was no significant difference in the mRNA expression of ephrinB2/EphB4/EphA2 between the two groups.10ng/ml,100ng/ml,and 1000ng/ml TNF-?,the expression of ephrinB2/EphB4 decreased and the expression of EphA2 increased(P<0.05).0.1ng/ml,1ng/ml concentration of TNF-? had no effect on ephrinB2/ EphB4 protein expression after 24 h,and 10ng/ml-1000ng/ml concentration decreased the expression of ephrinB2/EphB4,0.1ng/ml TNF-? increased the expression of EphB4 after 48 h,but ephrinB2 expression was not significantly changed after 1ng/ml,and the expression of ephrinB2/EphB4 decreased after 10ng/ml,1000ng/ml stimulation.3.The expression of EphB4 in the periodontitis group was significantly lower than that in the control group(p<0.01).Conclusion:1.Compared with other methods,the method of tissue block is easier to operate,but the success rate is lower,and the requirement of culture condition is higher.2.The expressions of ephrinB2,EphB4 and EphA2 changed with the change of TNF-? concentration over time,and the expressions of ephrinB2 /EphB4 were not all decreased under TNF-? effect.3.The decrease of EphB4 expression in gingival tissue of periodontitis patients may be related to the destruction of alveolar bone in periodontitis.