The Study On The Effect Of TNF-α On Angiogenesis In HUVECs And HDPSCs Co-culture System Through EphrinB2

Background and ObjectiveDental pulp tissue regeneration engineering is a promising method of necrotic or traumatic dental pulp treatment.Successful dental pulp tissue regeneration depends on the rapid development of local microvascular network,which can provide enough oxygen and nutrition for cells.Angiogenesis is a complex dynamic process regulated by a series of molecular and cellular interactions.It has been reported that both vascular endothelial growth factor(VEGF)family and Tyrosine kinase(Eph)family play an important role in angiogenesis.Among them,Eph-associated receptor tyrosine kinase ligand B2(Ephrin B2 ligand)and Receptor tyrosine kinase B4(Eph B4receptor)are specifically considered as key regulators in angiogenesis.They are membrane binding proteins,and Ephrin B2 can stabilize tubule formation.Tumor Necrosis Factor-alpha(TNF-α)is a marker of early inflammation,and low concentration of TNF-α can promote tubule formation.Therefore,the purpose of this study was to study the effect of Ephrin B2 on the tubular formation of umbilical vein endothelial cell(HUVECs)and human dental pulp stem cell(hDPSCs)under the action of TNF-α.MethodHUVECs and hDPSCs were co-cultured in vitro at a ratio of 5:1.The cells were treated with TNF-α(0-80 ng/m L),and the expression of Ephrin B2 protein was measured by Western blot.20 ng/m L TNF-α was used in the following experiments.To suppress the expression of Ephrin B2 in hDPSCs,Ephrin B2 sh RNA was transfected into hDPSCs,and then the relative length and node number of vascular-like structure was detected by tubule formation experiment in vitro.The expression of Ephrin B2,Eph B4 and Ephrin B2 sh RNA was observed by immunoflu-orescence.The expression of Ephrin B2,Eph B4 and VEGF was detected by Western blot.The experiment was divided into three groups: group a(TNF-α co-culture group):TNF-α+HUVECs+ hDPSCs,group b(TNF-α co-culture silence group):TNF-α+HUVECs+Ephrin B2 sh RNA-hDPSCs group,and group c(co-culture silence group): HUVECs+Ephrin B2 sh RNA-hDPSCs.Results(1)The result of western blot showed that TNF-α in of 20 ng/m L significantly promoted the expression of Ephrin B2 in co-culture system(P<0.001).(2)The results of tubule formation in vitro showed that at the 9,12 and 24 h of co-culture,the number of relative branches of tubules in the group c(co-culture silent)was less than that in the group a(TNF-α co-culture group)and group b(TNF-α co-culture silence group)(P<0.01),and the relative length was shorter than that in the group a and group b(P<0.01).(3)The results of immunofluorescence showed that red fluorescence(Ephin B2)and blue fluorescence(Eph B4)appeared almost in the same position in group a,and the merged image was pink.The green fluorescent region(silent hDPSCs)of group b and group c had neither red fluorescence nor blue fluorescence.Comparing the fluorescence intensity of the three groups,it was found that the overall expression levels of red fluorescence and blue fluorescence in group a were stronger than those in group b and group c(P<0.05).(4)Western blot results showed that compared with the group b,group a had higher expression of Ephrin B2,Eph B4 and VEGF proteins(P<0.01).Compared with the group c,the expression of Ephrin B2,Eph B4 and VEGF proteins in group b was higher(P<0.01).ConclusionIn the co-culture system of hDPSCs and HUVECs,silencing Ephrin B2 in hDPSCs can decrease the ability of tubule formation,and TNF-α(20 ng/m L)can activate the Ephrin B2-Eph B4 signal pathway in the co-culture system,save the division of tubules caused by silencing Ephrin B2,and promote the formation and stability of tubules.