目的：通过配制不同浓度的釉基质蛋白（enamel matrix proteins，EMPs）培养液，体外培养SD大鼠脂肪源性干细胞（adipose-derived stem cells, ADSCs），初步探究EMPs对ADSCs体外增殖活性及成骨分化能力的影响。方法：酶消化法获得大鼠脂肪间充质细胞，通过流式细胞仪技术分离、筛选干细胞，多向诱导分化对所得细胞进行鉴定；乙酸法制备EMPs干粉并通过蛋白免疫印记技术鉴定其成分，配制不同浓度的诱导液以备用；采用MTT法检测EMPs对ADSCs增殖活性的影响，并用实时荧光定量PCR及蛋白免疫印迹的方法检测EMPs诱导ADSCs成骨分化标志物的mRNA及蛋白表达变化情况。结果：釉基质蛋白（EMPs）对大鼠脂肪源性干细胞(ADSCs)的增殖具有明显的促进作用(P<0.05)，并呈现剂量和时间依赖性； EMPs培养ADSCs 14 d后，成骨分化标志物Runx-2、Osteocalcin(OCN)、Collagen (Col-I)在mRNA和蛋白水平表达均增加，并且与EMPs成正比（P<0.05）。结论：釉基质蛋白可作为一种成骨诱导剂，能有效增强大鼠脂肪源性干细胞体外增殖活性并诱导其成骨分化。%Objective: This study aimed to explore the impact of enamel matrix proteins (EMPs) on proliferation activity and osteogenic differentiation capacity of SD rat adipose-derived stem cells in vitro. Methods:Adipose-derived stem cells (ADSCs) were isolated from subcutaneous fat pads of healthy male SD rats aged 4 weeks by enzyme digestion method . Stem cells are separated and identified by flow cytometry technology. EMPs were extracted from tooth germs of heathy male pigs aged 4-6 months. SDS-PAGE electrophoresis technique was used to identify its characteristics, and frozen-dried powder were prepared for subsequent experiments. Effects of EMPs on cell proliferation were assessed by MTT assay. Ef-fects of EMPs on osteogenic differentiation of rat ADSCs were analyzed by Q-PCR and Western-Blot technique.Osteogenic differentiation markers of Osteocalcin (OCN), Runx-2, Collagen I (Col-I) were detected. Results: MTT assay showed that EMPs could enhance rat ADSC proliferation in a dose-dependent and time-dependent manner(P<0.05). Results of Q-PCR and Western-blot technique indicated that EMPs promoted expressions of Runx-2, Col-I and OCN at the mRNA level and protein level without osteo-induction medium (P<0.05). Conclusion: Enamel matrix proteins may be used as an osteoin-ductive agent, due its enhancing effect on adipose-derived stem cells proliferation and osteogenic differentiation.
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