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目的:研究釉基质蛋白(Enamel Matrix Proteins,EMPs)对大鼠牙髓干细胞(rat Dental Pulp Stem Cells,rDPSCs)体外增殖分化能力的影响。方法:酶消化培养法获得大鼠牙髓干细胞,有限稀释法分离纯化大鼠牙髓干细胞,形态学观察,计算细胞克隆形成率。乙酸法制备EMPs,用不同浓度的EMPs进行诱导,利用四唑盐比色法(MTT)检测和分析诱导后细胞增殖活性的变化。检测诱导后培养液中的碱性磷酸酶(Alkaline Phosphatase,ALP)。免疫组化染色和RT-PCR方法检测牙本质涎蛋白(dentin sialoprotein,DSP)、牙本质基质蛋白1(dentin matrixprotein1,DMP-1)和牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)的mRNA表达。结果:大鼠牙髓干细胞呈集落状生长,在体外具有一定的克隆形成能力。EMPs对大鼠牙髓干细胞的增殖有促进作用,并呈现剂量和时间依赖性。200μg/mlEMPs组可显著促进大鼠牙髓干细胞的增殖。EMPs作用组能显著提高大鼠牙髓干细胞的碱性磷酸酶活性。经细胞因子刺激后细胞表达DSP、DMP-1蛋白和DSPP mRNA。结论:EMPs在大鼠牙髓干细胞增殖和向成牙本质细胞分化方面具有积极的作用。
Objective: To investigate the effects of Enamel Matrix Proteins (EMPs) on proliferation and differentiation of rat dental pulp stem cells (rDPSCs) in vitro. METHODS: Rat dental pulp stem cells were obtained by enzymatic digestion and culture, and dental pulp stem cells were isolated and purified by limiting dilution. Morphology was observed and cell colony formation rate was calculated. EMPs were prepared by acetic acid method and induced by different concentrations of EMPs. MTT assay was used to detect and analyze the changes of cell proliferation after induction. Alkaline Phosphatase (ALP) in the culture medium was detected after induction. The expression of dentin sialoprotein (DSP), dentin matrix protein 1 (DMP-1) and dentin sialophosphoprotein (DSPP) mRNA were detected by immunohistochemistry and RT-PCR. Results: The dental pulp stem cells of rats showed colony-like growth and had some clonogenic capacity in vitro. EMPs can promote the proliferation of rat dental pulp stem cells in a dose-and time-dependent manner. 200μg / mlEMPs group can significantly promote the proliferation of rat dental pulp stem cells. EMPs group significantly increased alkaline phosphatase activity of rat dental pulp stem cells. Cells stimulated by cytokines expressed DSP, DMP-1 protein and DSPP mRNA. Conclusion: EMPs play a positive role in proliferation and differentiation of odontoblasts in rat dental pulp stem cells.