Effect Of Ectodysplasin-A1 Mutants On Biological Behaviors Of Odontogenic Cells

Objective:The aim of this study was to investigate the effects of transfected ectodysplasin-A,transcript variant 1?EDA1?mutants on the proliferation and cell cycle distribution of the ameloblast-like cells?LS8 cells?and dental pulp stem cells?DPSCs?,therefore to provide a theoretical basis for pathogenic mechanism of EDA1 mutation in tooth agenesis in further study.Methods:The cells were cultured in DMEM medium containing 10%fetal bovine serum,and cells were passaged with 0.25%trypsin+EDTA.Recombinant eukaryotic expression vectors EDA1-Wt?Wt group?,EDA1-A259E,EDA1-R334H,EDA1-S374R?NSTA groups?,EDA1-H252L?XLHED group?and pCR3?pCR3 group,control group?were transfected into LS8 cells and DPSCs.The MTT assay was performed to assess the viability of the cells.The effects of EDA1 mutants on cell cycle distribution of LS8 cells and DPSCs were detected by flow cytometry.SPSS statistical analysis software,version21.0 was used to conduct analysis of variance testing for all data.P<0.05 was considered to indicate a statistically significant difference.Results:1.The proliferation of LS8 cells was investigated by MTT assay.The LS8cells viability at 72 h infection increased significantly in the Wt group by comparison with the pCR3 group?P<0.05?.At 96h,the LS8 cells viability increased significantly in the Wt group by comparison with the XLHED group and the pCR3 group?P<0.05,P<0.01?,respectively.However,compared with the Wt group,the XLHED group and the pCR3 group,there was no significant difference in NSTA groups.2.Flow cytometric analysis was conducted to determine the effect of plasmid-mediated EDA1 transfection on the cell cycle distribution of LS8 cells.In terms of the percentage of LS8 at G₀/G₁ phase,the XLHED group was significantly higher compared with other four groups?wt group and NSTA groups??P<0.05?,respectively.During the S phase,the XLHED group showed significantly lower cell percentage than the other four groups?P<0.01?,respectively.3.The proliferation of DPSCs was investigated by MTT assay.The study found no significant difference on proliferation between the groups?P>0.05?.To determine the effect of the above EDA1 infection on the cell cycle distribution of DPSCs,flow cytometric analysis was conducted.It has been observed that there was no significant difference on the cell cycle distribution between the groups?P>0.05?.Conclusions:1.Compared with XLHED mutant EDA1,wild-type EDA1 promoted the proliferation of LS8 cells;2.Compared with wild-type EDA1 and NSTA mutant EDA1,XLHED mutant EDA1 blocked cell cycle progression of LS8 cells in the G₀/G₁ phase and decreased the proportion of LS8 cell cycle at S phase;3.However,no significant difference was found between wild-type EDA1,NSTA mutant EDA1 and XLHED mutant EDA1 in cell cycle distribution and proliferation of DPSCs,respectively.