Effects Of Dasatinib On The Proliferation And Osteogenesis Of Human Periodontal Ligament Stem Cells

Objective:This study was designed to investigate the effect of dasatinib on the proliferation,osteogenic differentiation,and possible regulatory mechanism at the molecular level of hPDLSCs.Methods:1.HPDLSCs were obtained by tissue block sticking method.The expression of MSCs markers was detected by flow cytometry.The clonal proliferation ability was examined by crystal violet staining.Above all were designed to determine whether they were mesenchymal originated stem cells.2.The differentiation ability of hPDLSCs induced by osteogenic medium was detected with alizarin red staining.The lipid droplets induced by adipogenic medium was observed by oil red O staining.Above all were designed to determine whether the cells were multiple differentiation potential.3.CCK-8 cell activity assay was used to evaluate how the dasatinib concentration working on the proliferation of hPDLSCs,with cytological microscope was employed to examine the morphological changes.The expression of proliferation and apoptosis related factors(Erk,Akt,Bcl2,etc.)were inspected by qRT-PCR and Western Blot.4.ALP staining and alizarin red staining were used to observe the effect of dasatinib concentration on the osteogenic differentiation ability of hPDLSCs.The expression of osteogenic related indicators(COL-1,RUNX2)were inspected by Western Blot.5.The expression of EID3 after osteogenic induction was detected by Western Blot.6.HPDLSCs were transfected with lentivirus to construct a model with EID3 specificly knockdown.The transfection was closely monitored under the fluorescence microscope,and the knockdown efficiency was appraised by Western Blot.7.the results of specific knockdown of EID3 on the osteogenic differentiation altering of hPDLSCs with dasatinib treatment were determined by ALP staining and alizarin red staining,with Western Blot was used to detect the expression of osteogenic related indicators(COL-1,RUNX2)on protein level.Results:1.The obtained cells were mesenchymal derived with excellent proliferative activity and multi-directional differentiation potential,which met the needs of follow-up researches.2.The effect of dasatinib on the proliferation of hPDLSCs was dose-dependent and time-dependent.When the concentration of dasatinib was more than 500nM,the proliferation of hPDLSCs was seriously damaged.3.Dasatinib has a concentration dependent effect on the osteogenic differentiation of hPDLSCs.The results showed that InM dasatinib had promotion on the osteogenic differentiation of hPDLSCs.However,when the dasatinib consistence was higher than 5nM,it turned to inhibit the protein expression of early osteogenic markers.A obvious declining change,which was observed by ALP staining and alizarin red staining,could be noticed when the concentration reached 10nM.4.EID3 participated in the osteogenic differentiation of hPDLSCs and played a negative regulatory role in it.After specific interference with EID3,the osteogenic differentiation of hPDLSCs were up-regulated.5.EID3 was involved in the osteogenic differentiation of of hPDLSCs regulated by high concentration of dasatinib(10nM).After specific knocked down EID3,The inhibitory effect of 10 nM dasatinib on osteogenesis was alleviated.Conclusion:The effect of dasatinib on the proliferation and osteogenic differentiation of hPDLSCs was concentration dependent.Low concentration of dasatinib can promote the osteogenic induction.EID3 negatively regulates the differentiation of hPDLSCs,and participate in the regulation of high concentration of dasatinib on the osteogenic inhibition of hPDLSCs.