miR-124对牙髓间充质干细胞成骨分化的影响及机制探讨

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

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5μmol/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-824、48、72 h A_(450)值,ALP活性A_(450)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-824、48、72 h A_(450)值、ALP活性A_(450)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。

PURPOSE:To evaluate the effect of microRNA(miR)-124 on osteogenic differentiation of dental pulp mes-enchymal 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-difluo-rophenacetyl)-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 5μmol/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 di-vision-related enhancer 1(HEY1),hair-like division-related enhancer 2(HEY2),and cyclin D1 gene(CCND1)were test-ed by Western blot.The data was analyzed by SPSS 19.0 software package.RESULTS:Compared with the blank group and no-load group,the A_(450) value at 24,48,72 h detected by CCK-8 experiment,A_(450) 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 A_(450) value at 24,48,72 h detected by CCK-8 experiment,A_(450) 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 ac-tion is related to the activation of Notch signaling pathway.