釉基质蛋白（EMPs）对大鼠牙髓干细胞体外增殖、分化能力的影响

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（MTD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 matrixprotein 1，DMP—1）和牙本质涎磷蛋白（dentin sialophosphopmtein，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。200gg／ml EMPs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。

Effects of EMPs on proliferation and differentiation of ratdental pulp stem cells in vitro

Objective： To investigate the effect of EMPs on the proliferation and differentiation of rat dental pulp stem cells in vitro. Method： Mono-cell suspension was separated from the rat dental pulp with collagenase I and dispase digestion. Colony-forming efficiency of cells was calculated. EMPs was made by acetic acid method, the dental pulp stem cells were affected by different concentrations of EMPs.The effect was examed by MTT assay.Alkaline Phosphatase, DSP, DMP-1 protein and DSPPmRNA were examed after inducement. Result： Clonogenic cells were obtained from dental pulp and the colony-rate is 1.8%.The proliferation of rDMSCs was significantly stimulated by EMPs.EMPs also increased the activity of ALP intracellular.DSP, DMP-1 protein and DSPPmRNA expressed in effected cells. Conclusion： EMPs can play a positive role in promoting the proliferation and differentiation of rDPSCs.