釉质基质蛋白通过miR-32对乳牙牙髓干细胞成骨与成脂分化的影响

目的: 探讨釉质基质蛋白（EMPs）对乳牙牙髓干细胞（SHED）成骨和成脂作用的影响,并探讨其分子机制。方法: 采用流式细胞术检测SHED表面抗原CD73、CD146、CD34和CD45的表达。通过OB成骨诱导液诱导SHED,采用茜素红染色检测其成骨分化能力。将SHED分为4组,NC组为无效序列shRNA干扰SHED,EMPs组为无效序列shRNA干扰SHED后,再用100 μg/L EMPs干预SHED,miR-32 inhibitor组为miR-32 shRNA质粒干扰SHED,EMPs+miR-32 inhibitor组为沉默miR-32后,再用100 μg/L的EMPs组干预SHED。qPCR检测miR-32、牙本质涎磷蛋白 (DSPP)、牙本质基质蛋白1 (DMP-1)、过氧化物酶体增殖剂激活受体γ（PPARγ）和CCAAT增强子结合蛋白α（C/EBPα）的基因表达。采用Western免疫印迹法检测DSPP、DMP-1、PPARγ和C/EBPα蛋白表达量,茜素红染色检测SHED成骨能力,油红O染色检测SHED成脂能力。采用GraphPad Prism 5软件进行统计学分析。结果: 流式细胞术结果显示,SHED的CD146和CD73阳性表达,CD34和CD45阴性表达,与干细胞表面标志物特征一致。茜素红染色和油红O染色显示,矿化结节和油滴明显增多,与干细胞多向分化特征一致。与NC组相比,EMPs组中miR-32基因表达量显著增加（P<0.05）,miR-32 inhibitor组和EMPs+miR-32 inhibitor组中miR-32表达量显著减低（P<0.05）。与NC组相比,EMPs组DSPP和DMP-1表达量,矿化结节数量显著增加（P<0.05）,PPARγ和C/EBPα表达量和油脂滴数量显著降低（P<0.05）,而miR-32 inhibitor组结果与之相反（P<0.05）。与miR-32 inhibitor组相比,EMPs+miR-32 inhibitor组DSPP、DMP-1、PPARγ和C/EBPα表达量,矿化结节数量和油脂滴数量无显著差异（P>0.05）。与EMPs组相比,EMPs+miR-32 inhibitor组DSPP和DMP-1表达量,矿化结节数量显著降低（P<0.05）,而PPARγ和C/EBPα表达量及油脂滴数量显著增加（P<0.05）。结论: EMPs通过促进miR-32的表达,从而调控SHED的成骨和成脂分化。

Effect of enamel matrix protein on osteogenic and adipogenic differentiation of dental pulp stem cells of deciduous teeth through miR-32

PURPOSE: To explore the effect of enamel matrix proteins(EMPs) on osteogenesis and adipogenesis of stem cells from human exfoliated deciduous teeth SHED), and explore its molecular mechanism. METHODS: SHEDs were used to detect the expression of its surface antigens CD73, CD146, CD34 and CD45 by flow cytometry. SHED was induced by OB osteogenic induction liquid, and then the osteogenic differentiation ability was measured by alizarin red staining. SHEDs were divided into 4 groups, NC group had invalid sequence shRNA interfered with SHED, EMPs group had invalid sequence shRNA interfered with SHED. Then 100 μg/L EMPs was used to interfere with SHED. In miR-32 inhibitor group, miR-32 shRNA plasmid was used to interfere with SHED; while in EMPs+miR-32 inhibitor group, 100 μg/L EMP was used to intervene SHED after silencing miR-32. QPCR was used to detect the expression of miR-32, dentin sialophosphoprotein (DSPP), dentin matrix protein 1, DMP-1, peroxisome proliferators-activated receptor γ (PPARγ) and CCAAT enhancer binding protein α (C/EBPα) gene expression; Western blot was used to detect the expression of DSPP, DMP-1, PPARγ and C/EBPα protein expression; Alizarin red staining was used to detect SHED osteogenic capacity; Oil red O staining was used to detect adipogenetic capacity of SHED. RESULTS: The results of flow cytometry showed that SHED had positive expression of CD146 and CD73, and negative expression of CD34 and CD45, which was consistent with the characteristics of stem cell surface markers. Alizarin red staining and oil red O staining showed mineralized nodules and oil droplets increased significantly, consistent with the multi-directional differentiation characteristics of stem cells. Compared with NC group, the expression of miR-32 gene in EMPs group was significantly increased(P<0.05), and the expression of miR-32 in miR-32 inhibitor group and EMPs+miR-32 inhibitor group was significantly decreased(P<0.05). Compared with NC group, the expression of DSPP and DMP-1, the number of mineralized nodules in EMPs group were significantly increased(P<0.05), the expression of PPARγ and C/EBPa and the number of lipid droplets were significantly decreased (P<0.05), while the result of miR-32 inhibitor group was the opposite (P<0.05). Compared with miR-32 inhibitor group, there was no significant difference in the expression of DSPP, DMP-1, PPARγ and C/EBPα, number of mineralized nodules and oil droplets in EMPs+miR-32 inhibitor group(P>0.05). Compared with EMPs group, the expression of DSPP and DMP-1 and the number of mineralized nodules in EMPs+miR-32 inhibitor group were significantly reduced(P<0.05), while the expression of PPARγ and C/EBPα and the number of lipid droplets were significantly increased(P<0.05). CONCLUSIONS: EMPs can regulate osteogenic and adipogenic differentiation of SHED by promoting the expression of miR-32.