The Role Of NFIC On CAMP-mediated Differentiation Of Stem Cells From The Apical Papilla

Objective Stem cells from apical papilla(SCAPs)are one of the dental mesenchymal stem cells residing in the root apex of developing permanent teeth which contributes to the formation of radicular pulp and root dentin.Signal transduction and transcription factors play an important role in the proliferation and differentiation of SCAPs.Researches had shown that c AMP signaling and NFIC promoted proliferation and odonto/osteogenic differentiation of SCAPs.In the current study,lentiviral vector that overexpressed NFIC gene was transfected into SCAPs to investigate the role of NFIC on the stimulation effects of c AMP-induced differentiation of SCAPs.Methods SCAPs isolated from dental papilla of human immature third molars were cultured by enzyme digestion.To identify the specific surface antigens of the cultured cells,flow cytometric analysis was performed.For multi-lineage differentiation study,the cells were induced and stained with Alizarin red S and oil red O.Western blot was used to detect transfection efficiency after lentiviral transfection.SCAPs transfected with lentivirus that overexpressed NFIC gene(ov-NFIC)or an empty vector(LV-empty)were co-treated with Forskolin.Mineralized nodule formation of each groups were measured by alizarin red staining and quantitative calcium analysis.Quantitative real-time reverse-transcription polymerase chain reaction(QPCR)were performed to test the expression of RUNX2,ALP and OCN m RNA.Results Flow cytometry indicated that SCAPs expressed mesenchymal stem cell(MSC)surface markers CD44,CD105 and CD90 positively,but negatively for thehematopoietic markers CD45.Alizarin red S and oil red O staining revealed that the isolated cells possessed the capability of odonto/osteogenic differentiation.After SCAPs were transfected with lentivirus overexpressing NFIC,a strong green fluorescence was observed under fluorescence microscope.Western blot results showed that the expression of NFIC protein were higher in ov-NFIC group compared with the control group significantly.After 10 days of mineralization induction,Alizarin red staining showed that Forskolin-treated SCAPs presented a notable rise in the number of mineralized nodules.Futhermore,when transfected with NFIC enhanced the promotion effects of Forskolin on SCAPs mineralization.QPCR were performed to test the expression of odonto/osteogenic markers which revealed that Forskolin increased the expression of RUNX2,ALP and OCN m RNA in SCAPs,and the stimulation effects of Forskolin were enhanced by overexpressing NFIC gene.Conclusion Our results indicated that SCAPs cultured by enzyme digestion were confirmed as mesenchymal stem cells by flow cytometry.NFIC could be involved in regulating the promotion effect of c AMP signaling on SCAPs.