釉基质蛋白对大鼠脂肪源性干细胞体外成骨分化能力的实验研究

Enamel Matrix Protein Mediates Rats Adipose-Derived Stem Cells Differentiation In Vitro

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m RNA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m RNA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.