| AimStem cells from the apical papilla (SCAPs) are a population of mesenchymal stem cells (MSCs) residing in root apex of developing teeth. SCAPs appear to have a capacity of multi-lineage differentiation. Compared with dental pulp stem cells (DPSCs), SCAPs show stronger proliferation and differentiation potential. In the process of tooth root development, SCAPs can differentiate into odontoblasts and form root dent in. However, molecules regulating SCAPs differentiation have not been elucidated. Zinc fingers and homeoboxes 2 (ZHX2) is a novel transcriptional inhibitor, ZHX2 mRNA widely expressed in multiple tissues. It is reported to modulate the development of nerve cells, liver cells, B cells, red blood cells, and so on. However, the role of ZHX2 in tooth root development remains unclear. Tooth formation is mediated by a serious of signal molecules. Whether ZHX2 participating in this process has not yet been reported. The purpose of this study is to reveal the potential role of ZHX2 in the proliferation and differentiation of SCAPs.Methods and results1. Isolution, Culture and identification of SCAPsEnzyme digestion was used to obtain SCAPs. In order to observe the osteo/odontogenic and adipogenic differentiation potency, SCAPs were treated by 4 weeks mineralization induction or 2 weeks adipogenic induction. Alizarin Red staining and Oil Red O staining showed the formation of mineralized nodules and lipid droplets; Immunofluorescence staining detected the high expression of STRO-1 In SCAPs; Furthermore, FCM analysis demonstrated SCAPs were positive for CD146 and CD24. Meanwhile, the expression of CD45 was negative.2. ZHX2 endogenous expression in SCAPs and the correlation between ZHX2 and osteo/odontogenic differentiationSCAPs were cultured by standard medium. RT-PCR and Western Blotting were conducted to detect ZHX2 endogenous expression in SCAPs, and results showed that ZHX2 was expressed in SCAPs; After mineralization induction with different time, the correlation between ZHX2 and osteo/odontogenic differentiation was tested by RT-PCR and Western Blotting. The results revealed that ZHX2 expression levels increased with the extension of mineralization time which indicated ZHX2 and mineralization were positively correlated relationship.3. The effection of ZHX2 on SCAPs proliferation and osteo/odontogenic differentiationAfter over-expression or knockdown of ZHX2, SCAPs were cultured by standard medium. CCK8 assays showed that ZHX2 inhibited SCAPs proliferation.SCAPs were induced 1 week with mineralization medium after ZHX2 over-expression. Then, DSPP, RUNX2, BSP and OCN expression levels were detected by qRT-PCR and Western Blotting. The results demonstrated that the mRNA and protein levels of DSPP, RUNX2, BSP and OCN were up-regulated after ZHX2 over-expression. At the same time, ALP activity assay was conducted. The results showed ALP activity was also up-regulated after over-expression.At the same time, after ZHX2 knockdown, ALP activity of mineralization induced SCAPs were detected. The results showed ALP activity was down-reg ulated; qRT-PCR and Western Blotting were used to test the expression of rele vant mineralization genes after 1 week mineralization induction. The results de monstrated that mRNA and protein levels of DSPP, RUNX2, BSP and OCN w ere down-regulated after ZHX2 knockdown; Meanwhile, the mineralized nodule s and quantitative detection of calcium ions were observed after 2 week miner alizatin induction. The corresponding number of mineralized nodules and calciu mions were decreased compared with control group.ConclutionZHX2 endogenous expression was exist in SCAPs, and the expression level s of ZHX2 increased with the extension of cell mineralization time; ZHX2 can inhibit proliferation of SCAPs; ZHX2 has a positive regulatory role in SCAPs osteo/odontogenic differentiation. |
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