Dynamics Expression On Runx2 Gene Profile During Osteogenesis In Stem Cells From Human Exfoliated Deciduous Teeth

Objective:To investigate their biological properties of mesenchymal stem cells, SHEDs were isolated and cultured in vitro.Runx2 gene profiles were observed dynamically by RT-PCR during SHEDs osteogenesis.Methods:According to expremental method from Miura et al[4], pulp tissues from 6-10-years-old children were obtained, digested by enzymes and cultured in vitro. Then isolated and purified by limited dilution. The population doubling, proliferation and morphologic characteristics of both the primary and passage cells were observed, and the cytohistological features of the primary cells were observed by HE staining. Colony forming unit were tested. Detection of cell surface antigen and screening used flow cytometry and irnmunofluorescence staining. The third passage cells were cultured in the adipogenic medium, Oil red O staining was conducted to test lipid droplets occurred. The third passage cells were cultured in the osteogenic medium (including 10 mmol/1 ascorbic acid phosphate,50 mg/1β-glycerophosphate and 1×10-8 mol/L dexamethasone) over a 21-day-period. Mineralized nodules were detected by Alizarin red staining. For detection of Runx2 mRNA expression dynamically semi-quantitative RT-PCR was performed every two days.Results:1.Obtained stem cells from human exfoliated deciduous teeth by enzyme digestion and limited dilution. In general, two days after culture, the primary cells became adherent to the plate and presented multiple forms. Then the polymorphism gradually reduced as the passages of cells. SHEDs showed a fibroblast-like morphology, with cell body enlarging, cell color darkening, and sizes and appearance becoming consistent. HE staining results showed that most of the cells presented fusiform shape with smaller size. The nucleus is round and large. A few cells contain more than one nucleolus. They could magnify themselves for 20 passages in vitro.2.The colony forming unit was 22-26/10(?) cells. Detection of cell surface antigen and screening used flow cytometry and immunofluorescence staining. The cells with CD146+account for 92.51±1.34% of total cells while the STRO-1+ accounts for 20.01±1.62%. Induce the cells to osteoblasts, which elicited biological and morphologic characteristics similar to those of osteoblasts and form the mineralized nodules in vitro. Alizarin red staining showed positive expression. Refraetion points of high intensity were showed in several cells after culture in medium with adipogenic induction and the cells are positive for oilred-O staining.3.The results of transcription factor Runx2 expression were depicted as a smooth line. Runx2 expression was up-regulated from day 0 to day 6 and subsequently dropped with an expression bottom at day 12, after that a second expression peak at day 18 occurred, followed by a stably regulation.ConcIusion:1.SHEDs could be isolated and cultured in vitro. SHEDs express surface markers of mesenchymal stem cells. SHEDs were identified to be a population of highly proliferativeand clonogenic cells capable of differentiating into adipocytes, and ostetoblasts.2.Runx2 gene profiles were observed dynamically during SHEDs osteoblastic differentiation. Runx2 express throughout every stage of osteoblastic differentiation. Runx2 expression was up-regulated during early and later stage and down-regulated in metaphase.