人脱落乳牙干细胞骨向分化相关分子Runx2的动态表达

Dynamic expression of Runx2 gene profile during osteogenesis in stem cells from human exfoliated deciduous teeth

背景:Runx2被认为是成骨基因表达的主要调节因子,是一种向成骨细胞分化的不可或缺的转移因子,在成骨细胞发育、分化、调控、骨钙化形成及骨修复过程中起着极其重要的作用。目的:观察人脱落乳牙干细胞生物学特征,探讨乳牙干细胞骨向分化潜能,以及不同时间点成骨相关转录因子Runx2的动态表达规律。方法:体外分离、培养人脱落乳牙干细胞。流式细胞仪检测乳牙干细胞表面标志。体外脂向诱导分化4周后进行油红O染色,检测脂滴形成情况;矿化诱导21 d进行茜素红染色,检测矿化结节形成情况。于成骨诱导的不同时间点,用RT-PCR技术检测Runx2的动态表达。结果与结论:通过酶消化法和有限稀释法获得了乳牙干细胞,流式细胞仪检测显示CD146和STRO-1均有不同程度的表达。成脂诱导油红O染色可见橙红色阳性颗粒。矿化诱导茜素红染色呈阳性。RT-PCR技术检测Runx2在成骨诱导第0天已有表达,0-6 d成明显增强趋势,6-12 d显著下降,12-18 d表达再次上升,以后相对平稳。结果表明乳牙干细胞可体外分离、培养,表达间充质干细胞表面标志,具有成脂和成骨向分化能力。Runx2在乳牙干细胞成骨分化各阶段均有表达,早期和晚期表达上升,中期表达有下降趋势。

BACKGROUND：Runx2 is considered to the main regulatory factor of osteogenic gene expression and be necessary for osteoblast differentiation, it plays an extremely important role in the osteoblast development, differentiation, regulation, bone calcification formation and bone repair. OBJECTIVE：To observe the biological properties of mesenchymal stem cells from human exfoliated deciduous teeth, explore the osteogenic differentiation potential of deciduous teeth stem cells, and observe the dynamic expression of Runx2 gene at varying time points. METHODS：The stem cells from human exfoliated deciduous teeth were isolated and cultured in vitro. The cellsurface antigen was detected with flow cytometry. The third passage cells were cultured in the adipogenic medium for 4 weeks, and oil red O staining was conducted to test lipid droplets formation. The third passage cells were cultured in the osteogenic medium for 21 days, and mineralized nodules were detected by alizarin red staining. Runx2 mRNA dynamic expression was detected with semi-quantitative RT-PCR at different time points. RESULTS AND CONCLUSION：The stem cells from human exfoliated deciduous teeth were obtained by enzyme digestion and limited dilution methods. Flow cytometry results showed that, CD146 and STRO-1 were expressed to varying degrees. Oil red O staining revealed salmon pink positive particles. Alizarin red staining showed positive expression. RT-PCR results showed that, Runx2 expression was found at day 0, up-regulated from day 0 to day 6, and subsequently dropped with an expression bottom at day 12, after that a second expression peak occurred at day 18, fol owed by a stably regulation. The stem cells from human exfoliated deciduous teeth can be isolated and cultured in vitro, express surface antigen of mesenchymal stem cells, and have the potentials of differentiating into adipocytes and ostetoblasts. Runx2 gene profiles are dynamical y expressed during osteoblastic differentiation. Runx2 express throughout every stage of osteoblastic differentiation. The expression is up-regulated during early and later stages, and down-regulated in metaphase.