miR-124对牙髓间充质干细胞成骨分化的影响及机制探讨

目的: 观察微小RNA（miR）-124对牙髓间充质干细胞（DPSCs）成骨分化的影响,并探讨其可能机制。方法: 取对数期DPSCs,分为空白组、空载组、miR-124 inhibitor组和miR-124 inhibitor联合氮-[氮-(3,5-二氟苯乙酰)-L-丙氨酰]-S-苯基甘氨酸丁酯（DAPT,Notch信号通路抑制剂]组。空白组不处理,空载组转染阴性对照载体inhibitor-NC,miR-124 inhibitor组转染miR-124 inhibitor,miR-124 inhibitor联合DAPT组转染miR-124 inhibitor,并加入DAPT使其终浓度为5 μmol/L。转染48 h后,采用CCK-8法检测增殖能力;经诱导液诱导2周后,采用对-硝基苯磷酸盐（P-NPP）法检测碱性磷酸酶（ALP）活性;茜素红染色法检测钙化结节面积;Western印迹法检测细胞中发状分裂相关增强子1（HEY1）、发状分裂相关增强子2（HEY2）和细胞周期蛋白D1基因（CCND1）蛋白表达量。采用SPSS 19.0软件包对数据进行统计学分析。结果: 与空白组、空载组相比,miR-124 inhibitor组CCK-8 24、48、72 h A₄₅₀值,ALP活性A₄₅₀值,钙化结节面积构成比和HEY1、HEY2、CCND1蛋白表达量显著升高（P<0.05）;与miR-124 inhibitor组相比,miR-124 inhibitor联合DAPT组CCK-8 24、48、72 h A₄₅₀值、ALP活性A₄₅₀值、钙化结节面积构成比和HEY1、HEY2、CCND1蛋白表达量显著降低（P<0.05）。结论: 下调miR-124可促进DPSCs成骨分化,推测其作用机制与激活Notch信号通路有关。

Effect,mechanism of miR-124on osteogenic differentiation of dental pulp mesenchymal stem cells

PURPOSE: To evaluate the effect of microRNA (miR)-124 on osteogenic differentiation of dental pulp mesenchymal stem cells (DPSCs) and to explore the possible mechanism. METHODS: Logarithmic DPSCs were collected and divided into blank group, no-load group, miR-124 inhibitor group, miR-124 inhibitor combined with N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-ph (DAPT, Notch signaling pathway inhibitor) group. The blank group was not treated, the empty group was transfected with negative control vector inhibitor-NC, the miR-124 inhibitor group was transfected with miR-124 inhibitor, the miR-124 inhibitor combined with DAPT group was transfected with miR-124 inhibitor, and DAPT was added to make the final concentration of 5 μmol/L. The proliferation ability was tested by CCK-8 method 48 h after transfection. Alkaline phosphatase (ALP) activity was tested by p-nitrophenyl phosphate (P-NPP) method after 2 weeks of induction. The area of calcified nodules was tested by alizarin red staining method. The protein expression of hair-like division-related enhancer 1 (HEY1), hair-like division-related enhancer 2 (HEY2), and cyclin D1 gene (CCND1) were tested by Western blot. The data was analyzed by SPSS 19.0 software package. RESULTS: Compared with the blank group and no-load group, the A₄₅₀ value at 24, 48, 72 h detected by CCK-8 experiment, A₄₅₀ value of ALP activity, the area composition ratio of calcified nodules, and expression of HEY1, HEY2, and CCND1 in the miR-124 inhibitor group were increased (P<0.05). Compared with miR-124 inhibitor group, the A₄₅₀ value at 24, 48, 72 h detected by CCK-8 experiment, A₄₅₀ value of ALP activity, the area composition ratio of calcified nodules, and the expression of HEY1, HEY2, and CCND1 in the miR-124 inhibitor combined with DAPT group were significantly decreased(P<0.05). CONCLUSIONS: Down-regulation of miR-124 can promote osteogenic differentiation of DPSCs. It is speculated that the mechanism of action is related to the activation of Notch signaling pathway.