Effects Of Endothelin B Receptor Agonist On Biological Behaviors Of Cementoblasts

1.Culture of dental cementoblastsObjective:To establish a cementoblast cell line,observe cell growth,test the mineralization ability of cells in vitro,and detect the presence of ETBR.Methods:The rat dental cement cell line OCCM-30 was cultured,the growth state was observed,the growth curve was drawn,the mineralization ability was detected by alizarin red staining,and the intracellular ETBR was detected by immunofluorescence.Results:The results of alizarin red assay showed that OCCM-30 can be induced mineralized nodules in vitro.The results of immunofluorescence showed that ETBR existed in cementum cells.Conclusion:OCCM-30 cells have in vitro differentiation potential,and there are ETBR in cells,which can be continued in subsequent experiments.2.Effect of endothelin B receptor agonist on proliferation,apoptosis and ALP of cementoblastsObjective:To investigate the effects of ETBR agonists on proliferation,apoptosis and ALP of OCCM-30 cell line.Methods:ETBR agonists with 10-9mol/L,10-8mol/L,10-7mol/L and 10-6mol/L were used as the experimental group,0 mol/L ETBR agonist was used as the control group,and MTT was used to detect the cement.Changes in cytoplasmic proliferation;flow cytometry to detect changes in apoptosis;alkaline phosphatase staining to detect changes in activity of cementitious cells.Results:MTT and alkaline phosphatase staining showed that ETBR agonists inhibited cell proliferation and cell viability and showed dose-dependentness within a certain range;Analysis of the results showed that ETBR agonists promoted early apoptosis and had no significant effect on late apoptosis.Conclusion:The inhibitory effect of ETBR agonists on the proliferation and cell viability of cementoblasts is positively correlated with the concentration of ETBR agonists,which promotes early apoptosis.3.Effect of endothelin B receptor agonist on the differentiation of cementoblasts.Objective:To investigate the effects of 10-9mol/L ETBR agonists on the mineralization and differentiation of OCCM-30 cells in vitro.Methods:Alizarin red staining was used to detect the mineralization of cells in vitro.Real-time PCR was used to detect the relative expression of Runx2,Bsp,Osterix,Ocn and Coll mRNA.The expression of Runx2 and Osterix was detected by Western blot.Results:Alizarin red staining showed that ETBR agonists inhibited cell mineralization in vitro;real-time PCR results showed prolonged action time,and the expression levels of Runx2,Bsp,Osterix,Ocn and Coll were down-regulated to varying degrees;Western blot showed Runx2 and Osterix expression down.Conclusion:10-9mol/L ETBR agonists can inhibit the mineralization and differentiation of OCCM-30 cells in vitro by down-regulating the relative expression levels of inhibit the differentiation-related genes Runx2,Bsp,Osterix,Ocn,Coll and differentiation-related proteins Runx2 and Osterix.